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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
Chemical composition, antibacterial and anticancer activities of volatile oil of Melicope denhamii leaves a
a
b
Sony George , S Ajikumaran Nair , Ramaswamy Venkataraman & a
Sabulal Baby a
Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, PachaPalode, Thiruvananthapuram 695 562, Kerala, India b
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Department of Chemistry, Sri Paramakalyani College (Manonmaniam Sundaranar University, Tirunelveli), Alwarkurichi 627 412, Tamil Nadu, India Published online: 23 Feb 2015.
To cite this article: Sony George, S Ajikumaran Nair, Ramaswamy Venkataraman & Sabulal Baby (2015): Chemical composition, antibacterial and anticancer activities of volatile oil of Melicope denhamii leaves, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1013471 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1013471
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,
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1013471
SHORT COMMUNICATION Chemical composition, antibacterial and anticancer activities of volatile oil of Melicope denhamii leaves Sony Georgea, S Ajikumaran Naira, Ramaswamy Venkataramanb and Sabulal Babya* a
Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, Kerala, India; bDepartment of Chemistry, Sri Paramakalyani College (Manonmaniam Sundaranar University, Tirunelveli), Alwarkurichi 627 412, Tamil Nadu, India
Downloaded by [FU Berlin] at 20:33 11 May 2015
(Received 19 December 2014; final version received 27 January 2015)
Melicope denhamii leaf volatile oil was isolated by hydrodistillation, and twenty-six constituents comprising 95.95% of the leaf oil were characterised by gas chromatographic techniques. Sesquiterpenes, zierone (22.49%) and a-gurjunene (19.96%), were identified as the major components. M. denhamii leaf oil tested against Gram-positive and Gram-negative bacteria showed significant activity against Bacillus subtilis and Escherichia coli. Anticancer activity of M. denhamii leaf oil against Dalton’s lymphoma ascites cells was assessed by trypan blue exclusion and MTT assays, and the oil showed significant cytotoxicity at CD50 of 12.2 mg/mL. Induction of apoptosis on DLA cells by M. denhamii leaf oil was confirmed by morphological observation, nuclear damage and comet assays. Keywords: Melicope denhamii; leaf oil; antibacterial; anticancer; MTT assay
1. Introduction Melicope denhamii (Seem.) T.G. Hartley (synonym: Euodia ridleyi Hochr.) is an evergreen medicinal shrub belonging to the family Rutaceae. It is widely distributed in Borneo, Philippines, India and the Solomon Islands (Nakashima, Oyama, Ito, Akao, et al. 2012). M. denhamii leaves are traditionally used against skin diseases including benign warts and as larvicidal agents (Prathibha et al. 2010; Nakashima, Oyama, Ito, Akao, et al. 2012). Recent phytochemical studies on the leaves of M. denhamii reported the isolation of several alkaloids, chromenes, coumarins and sesquiterpenes (Nakashima et al. 2011; Nakashima, Oyama, Ito, Akao, et al. 2012; Nakashima, Oyama, Ito, Witono, et al. 2012). In this study, we report the chemical profiling, antibacterial and anticancer activities of the leaf volatile oil of M. denhamii from south India.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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S. George et al.
2. Results and discussion 2.1. Chemical constituents of M. denhamii leaf oil
2.2. Antibacterial activity of the leaf oil M. denhamii leaf oil at 1:1 dilution when compared with streptomycin standard at 2 mg/disc showed significant antibacterial activity against Bacillus subtilis (MTCC No. 441, 18/21 mm) and Escherichia coli (MTCC No. 443, 9/10 mm) (Table S2). Compared with the antibacterial studies on other essential oils (Sabulal et al. 2006; Sabulal and George 2009), M. denhamii leaf oil only exerted antibacterial activity against a narrow spectrum of bacteria. 2.3. Cytotoxicity of the leaf oil Preliminary cytotoxicity (Table S3) of M. denhamii leaf oil tested by trypan blue exclusion assay showed concentration dependent cytotoxicity on DLA cells with CD50 10.9 mg/mL. We also conducted MTT assay to determine the cytotoxic effect of the leaf oil and it exhibited significant DLA cell death in a concentration dependent manner (CD50 12.2 mg/mL) (Figure 1). Vincristine, a standard anticancer drug, also showed significant anti-DLA activity with CD50 , 5 mg/mL. As given in Table S4, at 25 mg/mL, the leaf oil exhibited lesser cytotoxicity to macrophages, thymocytes and bone marrow cells compared with vincristine at the same dose. Induction of apoptosis is the characteristic of anticancer activity of plant molecules. Here, the apoptotic induction of M. denhamii leaf oil on DLA cells was determined by observing membrane blebbing, nuclear condensation and nuclear DNA damage. In fluorescent microscopy
120
Leaf oil
Vincristine
100 % Cell deatn
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Hydrodistillation of M. denhamii leaves (250 g) afforded 0.7 mL (0.28% v/w, fresh weight basis) clear, golden-yellow coloured oil. The physical parameters of leaf oil of M. denhamii were 20 20 recorded as n20 ¼ 0.9307. Twenty-six compounds D ¼ 1.5122, ½aD ¼ 2 38.09308 and d comprising 95.95% of the oil were characterised by GC-FID, GC-MS, mass spectral match and comparison of linear retention indices (Table S1). Major compound zierone (22.49%) was isolated by column chromatography of M. denhamii leaf oil and identified by 1H NMR, mass spectra, LRI, Adams index (Adams 2007) and co-GC. 1H NMR (400 MHz, CDCl3): d1.60 (s, 12Me), 2.06 (s, 13-Me), 1.01 (s, 14-Me), 1.57 (s, 15-Me) of zierone (Figure S1) isolated from the leaf oil was in good agreement with the literature data (Nakashima, Oyama, Ito, Witono, et al. 2012). a-Gurjunene (19.96%) was identified by Co-GC with its authentic standard (Figure S1).
80 60 40 20 0 5
10 25 Concentration (µg/mL)
50
100
Figure 1. In vitro cytotoxicity of M. denhamii leaf oil on DLA cells determined by MTT assay. Values are mean ^ SD of three separate determinations. DLA cells were incubated at 378C for 48 h in RPMI media in CO2 incubator. The control (0.01% DMSO) showed 0% cell death.
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(Figure S2(a) –(c)), the DLA cells treated with 25 mg/mL of M. denhamii leaf oil or vincristine for 48 h and stained with acridine orange-ethidium bromide showed loss of membrane integrity, membrane blebbing and appeared as yellowish-red colour (dead cells). In phase contrast microscopy (Figure S2(d) –(f)), leaf oil treated DLA cells showed nuclear condensation and membrane blebbing. Nuclear damage induced by leaf oil during apoptosis was assessed by comet assay, where the comet consisted of a head having nuclear matrix and a tail of DNA fragments formed during the last stage of apoptotic cascade. As shown in the Figure S2(g)– (i), the leaf oil and vincristine at 25 mg/mL induced DNA damage and loss in nuclear integrity as observed from the comet like appearance of the nuclear DNA. The significant anti-DLA activity shown by M. denhamii leaf oil may be attributed to the presence of high percentage of sesquiterpenes in the leaf oil (51.97% oxygenated sesquiterpenes and 40.10% sesquiterpene hydrocarbons). When compared with the activities of other sesquiterpene rich essential oils reported by Sylvestre et al. (2005) and Silva et al. (2008), M. denhamii leaf oil is highly active with a low CD50 of 12.2 mg/mL. Moreover, the presence of cytotoxic constituents such as b-elemene, E-caryophyllene and spathulenol may also have triggered the activity in the leaf oil. Even though the major compounds in the leaf oil, zierone and a-gurjunene, have no previous cytotoxic activities reported, the leaf oil still showed strong cytotoxic activity, which may be due to the synergetic effect shown by the major and minor components of the oil. 3. Conclusions In the present study, we reported the chemical profile and bioactivities of essential oil from M. denhamii leaves. The ability of M. denhamii leaf oil to induce apoptosis on DLA cells was confirmed from the morphological observations and nuclear damage by comet assay. Supplementary material Experimental details relating to this article are available online, alongside Tables S1– S4 and Figures S1 and S2. Acknowledgements This study was supported by a Plan Project Scheme of the Government of Kerala. S.G. is a doctoral student registered at MS University, Tirunelveli.
References Adams RP. 2007. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 4th ed. Carol Stream (IL): Allured Publishing Corporation. Nakashima KI, Oyama M, Ito T, Akao Y, Witono JR, Darnaedi D, Tanaka T, Murata J, Iinuma M. 2012. Novel quinolinone alkaloids bearing a lignoid moiety and related constituents in the leaves of Melicope denhamii. Tetrahedron. 68:2421–2428. doi:10.1016/j.tet.2012.01.007. Nakashima KI, Oyama M, Ito T, Witono JR, Darnaedi D, Tanaka T, Murata J, Iinuma M. 2011. Melicodenines A and B, novel diels– alder type adducts isolated from Melicope denhamii. Tetrahedron Lett. 52:4694–4696. doi:10.1016/ j.tetlet.2011.07.013. Nakashima KI, Oyama M, Ito T, Witono JR, Darnaedi D, Tanaka T, Murata J, Iinuma M. 2012. Novel zierane- and guaiane-type sesquiterpenes from the root of Melicope denhamii. Chem Biodivers. 9:2195–2202. doi:10.1002/ cbdv.201100345. Prathibha KP, Raghavendra BS, Vijayan VA. 2010. Evaluation of larvicidal effect of Euodia ridleyi Hochr. leaf extract against three mosquito species at Mysore. Res J Biol Sci. 5:452–455. doi:10.3923/rjbsci.2010.452.455. Sabulal B, George V. 2009. Essential oils and new antimicrobial strategies. In: Ahmad I, Aqil F, editors. New strategies combating bacterial infection. Weinheim, Germany: Wiley; p. 165–203.
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Sabulal B, George V, Shiburaj S. 2006. Volatile constituents and antibacterial activity of the flower oil of Evodia lunuankenda (Gaertn) Merr. J Essent Oil Res. 18:462–464. doi:10.1080/10412905.2006.9699141. Silva SLD, Chaar JDS, Figueiredo PDMS, Yano T. 2008. Cytotoxic evaluation of essential oil from Casearia sylvestris Sw on human cancer cells and erythrocytes. Acta Amaz. 38:107–112. doi:10.1590/S0044-59672008000100012. Sylvestre M, Legault J, Dufour D, Pichette A. 2005. Chemical composition and anticancer activity of leaf essential oil of Myrica gale L. Phytomedicine. 12:299–304. doi:10.1016/j.phymed.2003.12.004.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Chemical composition, antibacterial and anticancer activities of volatile oil of Melicope denhamii leaves a
a
b
Sony George , S Ajikumaran Nair , Ramaswamy Venkataraman & a
Sabulal Baby a
Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, PachaPalode, Thiruvananthapuram 695 562, Kerala, India b
Click for updates
Department of Chemistry, Sri Paramakalyani College (Manonmaniam Sundaranar University, Tirunelveli), Alwarkurichi 627 412, Tamil Nadu, India Published online: 23 Feb 2015.
To cite this article: Sony George, S Ajikumaran Nair, Ramaswamy Venkataraman & Sabulal Baby (2015): Chemical composition, antibacterial and anticancer activities of volatile oil of Melicope denhamii leaves, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1013471 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1013471
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 [FU Berlin] at 20:33 11 May 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/10.1080/14786419.2015.1013471
SHORT COMMUNICATION Chemical composition, antibacterial and anticancer activities of volatile oil of Melicope denhamii leaves Sony Georgea, S Ajikumaran Naira, Ramaswamy Venkataramanb and Sabulal Babya* a
Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode, Thiruvananthapuram 695 562, Kerala, India; bDepartment of Chemistry, Sri Paramakalyani College (Manonmaniam Sundaranar University, Tirunelveli), Alwarkurichi 627 412, Tamil Nadu, India
Downloaded by [FU Berlin] at 20:33 11 May 2015
(Received 19 December 2014; final version received 27 January 2015)
Melicope denhamii leaf volatile oil was isolated by hydrodistillation, and twenty-six constituents comprising 95.95% of the leaf oil were characterised by gas chromatographic techniques. Sesquiterpenes, zierone (22.49%) and a-gurjunene (19.96%), were identified as the major components. M. denhamii leaf oil tested against Gram-positive and Gram-negative bacteria showed significant activity against Bacillus subtilis and Escherichia coli. Anticancer activity of M. denhamii leaf oil against Dalton’s lymphoma ascites cells was assessed by trypan blue exclusion and MTT assays, and the oil showed significant cytotoxicity at CD50 of 12.2 mg/mL. Induction of apoptosis on DLA cells by M. denhamii leaf oil was confirmed by morphological observation, nuclear damage and comet assays. Keywords: Melicope denhamii; leaf oil; antibacterial; anticancer; MTT assay
1. Introduction Melicope denhamii (Seem.) T.G. Hartley (synonym: Euodia ridleyi Hochr.) is an evergreen medicinal shrub belonging to the family Rutaceae. It is widely distributed in Borneo, Philippines, India and the Solomon Islands (Nakashima, Oyama, Ito, Akao, et al. 2012). M. denhamii leaves are traditionally used against skin diseases including benign warts and as larvicidal agents (Prathibha et al. 2010; Nakashima, Oyama, Ito, Akao, et al. 2012). Recent phytochemical studies on the leaves of M. denhamii reported the isolation of several alkaloids, chromenes, coumarins and sesquiterpenes (Nakashima et al. 2011; Nakashima, Oyama, Ito, Akao, et al. 2012; Nakashima, Oyama, Ito, Witono, et al. 2012). In this study, we report the chemical profiling, antibacterial and anticancer activities of the leaf volatile oil of M. denhamii from south India.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
S. George et al.
2. Results and discussion 2.1. Chemical constituents of M. denhamii leaf oil
2.2. Antibacterial activity of the leaf oil M. denhamii leaf oil at 1:1 dilution when compared with streptomycin standard at 2 mg/disc showed significant antibacterial activity against Bacillus subtilis (MTCC No. 441, 18/21 mm) and Escherichia coli (MTCC No. 443, 9/10 mm) (Table S2). Compared with the antibacterial studies on other essential oils (Sabulal et al. 2006; Sabulal and George 2009), M. denhamii leaf oil only exerted antibacterial activity against a narrow spectrum of bacteria. 2.3. Cytotoxicity of the leaf oil Preliminary cytotoxicity (Table S3) of M. denhamii leaf oil tested by trypan blue exclusion assay showed concentration dependent cytotoxicity on DLA cells with CD50 10.9 mg/mL. We also conducted MTT assay to determine the cytotoxic effect of the leaf oil and it exhibited significant DLA cell death in a concentration dependent manner (CD50 12.2 mg/mL) (Figure 1). Vincristine, a standard anticancer drug, also showed significant anti-DLA activity with CD50 , 5 mg/mL. As given in Table S4, at 25 mg/mL, the leaf oil exhibited lesser cytotoxicity to macrophages, thymocytes and bone marrow cells compared with vincristine at the same dose. Induction of apoptosis is the characteristic of anticancer activity of plant molecules. Here, the apoptotic induction of M. denhamii leaf oil on DLA cells was determined by observing membrane blebbing, nuclear condensation and nuclear DNA damage. In fluorescent microscopy
120
Leaf oil
Vincristine
100 % Cell deatn
Downloaded by [FU Berlin] at 20:33 11 May 2015
Hydrodistillation of M. denhamii leaves (250 g) afforded 0.7 mL (0.28% v/w, fresh weight basis) clear, golden-yellow coloured oil. The physical parameters of leaf oil of M. denhamii were 20 20 recorded as n20 ¼ 0.9307. Twenty-six compounds D ¼ 1.5122, ½aD ¼ 2 38.09308 and d comprising 95.95% of the oil were characterised by GC-FID, GC-MS, mass spectral match and comparison of linear retention indices (Table S1). Major compound zierone (22.49%) was isolated by column chromatography of M. denhamii leaf oil and identified by 1H NMR, mass spectra, LRI, Adams index (Adams 2007) and co-GC. 1H NMR (400 MHz, CDCl3): d1.60 (s, 12Me), 2.06 (s, 13-Me), 1.01 (s, 14-Me), 1.57 (s, 15-Me) of zierone (Figure S1) isolated from the leaf oil was in good agreement with the literature data (Nakashima, Oyama, Ito, Witono, et al. 2012). a-Gurjunene (19.96%) was identified by Co-GC with its authentic standard (Figure S1).
80 60 40 20 0 5
10 25 Concentration (µg/mL)
50
100
Figure 1. In vitro cytotoxicity of M. denhamii leaf oil on DLA cells determined by MTT assay. Values are mean ^ SD of three separate determinations. DLA cells were incubated at 378C for 48 h in RPMI media in CO2 incubator. The control (0.01% DMSO) showed 0% cell death.
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(Figure S2(a) –(c)), the DLA cells treated with 25 mg/mL of M. denhamii leaf oil or vincristine for 48 h and stained with acridine orange-ethidium bromide showed loss of membrane integrity, membrane blebbing and appeared as yellowish-red colour (dead cells). In phase contrast microscopy (Figure S2(d) –(f)), leaf oil treated DLA cells showed nuclear condensation and membrane blebbing. Nuclear damage induced by leaf oil during apoptosis was assessed by comet assay, where the comet consisted of a head having nuclear matrix and a tail of DNA fragments formed during the last stage of apoptotic cascade. As shown in the Figure S2(g)– (i), the leaf oil and vincristine at 25 mg/mL induced DNA damage and loss in nuclear integrity as observed from the comet like appearance of the nuclear DNA. The significant anti-DLA activity shown by M. denhamii leaf oil may be attributed to the presence of high percentage of sesquiterpenes in the leaf oil (51.97% oxygenated sesquiterpenes and 40.10% sesquiterpene hydrocarbons). When compared with the activities of other sesquiterpene rich essential oils reported by Sylvestre et al. (2005) and Silva et al. (2008), M. denhamii leaf oil is highly active with a low CD50 of 12.2 mg/mL. Moreover, the presence of cytotoxic constituents such as b-elemene, E-caryophyllene and spathulenol may also have triggered the activity in the leaf oil. Even though the major compounds in the leaf oil, zierone and a-gurjunene, have no previous cytotoxic activities reported, the leaf oil still showed strong cytotoxic activity, which may be due to the synergetic effect shown by the major and minor components of the oil. 3. Conclusions In the present study, we reported the chemical profile and bioactivities of essential oil from M. denhamii leaves. The ability of M. denhamii leaf oil to induce apoptosis on DLA cells was confirmed from the morphological observations and nuclear damage by comet assay. Supplementary material Experimental details relating to this article are available online, alongside Tables S1– S4 and Figures S1 and S2. Acknowledgements This study was supported by a Plan Project Scheme of the Government of Kerala. S.G. is a doctoral student registered at MS University, Tirunelveli.
References Adams RP. 2007. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 4th ed. Carol Stream (IL): Allured Publishing Corporation. Nakashima KI, Oyama M, Ito T, Akao Y, Witono JR, Darnaedi D, Tanaka T, Murata J, Iinuma M. 2012. Novel quinolinone alkaloids bearing a lignoid moiety and related constituents in the leaves of Melicope denhamii. Tetrahedron. 68:2421–2428. doi:10.1016/j.tet.2012.01.007. Nakashima KI, Oyama M, Ito T, Witono JR, Darnaedi D, Tanaka T, Murata J, Iinuma M. 2011. Melicodenines A and B, novel diels– alder type adducts isolated from Melicope denhamii. Tetrahedron Lett. 52:4694–4696. doi:10.1016/ j.tetlet.2011.07.013. Nakashima KI, Oyama M, Ito T, Witono JR, Darnaedi D, Tanaka T, Murata J, Iinuma M. 2012. Novel zierane- and guaiane-type sesquiterpenes from the root of Melicope denhamii. Chem Biodivers. 9:2195–2202. doi:10.1002/ cbdv.201100345. Prathibha KP, Raghavendra BS, Vijayan VA. 2010. Evaluation of larvicidal effect of Euodia ridleyi Hochr. leaf extract against three mosquito species at Mysore. Res J Biol Sci. 5:452–455. doi:10.3923/rjbsci.2010.452.455. Sabulal B, George V. 2009. Essential oils and new antimicrobial strategies. In: Ahmad I, Aqil F, editors. New strategies combating bacterial infection. Weinheim, Germany: Wiley; p. 165–203.
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Downloaded by [FU Berlin] at 20:33 11 May 2015
Sabulal B, George V, Shiburaj S. 2006. Volatile constituents and antibacterial activity of the flower oil of Evodia lunuankenda (Gaertn) Merr. J Essent Oil Res. 18:462–464. doi:10.1080/10412905.2006.9699141. Silva SLD, Chaar JDS, Figueiredo PDMS, Yano T. 2008. Cytotoxic evaluation of essential oil from Casearia sylvestris Sw on human cancer cells and erythrocytes. Acta Amaz. 38:107–112. doi:10.1590/S0044-59672008000100012. Sylvestre M, Legault J, Dufour D, Pichette A. 2005. Chemical composition and anticancer activity of leaf essential oil of Myrica gale L. Phytomedicine. 12:299–304. doi:10.1016/j.phymed.2003.12.004.
