Isolation of Antibacterial Compounds from Artemisia ...
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Abstract The Chumash Na+ve Americans of Southern California have well documented tradi+ons of using plants for medicinal purposes. If a specific plant has tradi+onally been used by Chumash for the treatment of cuts, wounds and infec+ons, it may contain chemicals with an+bacterial proper+es. One plant that fits these criteria is Artemisia californica (coastal sage). Because of the widespread use of an+bio+cs over the past sixty years bacteria are evolving greater resistance to known an+bio+cs, but unfortunately the rate of an+bio+c discovery has diminished during the past twenty years. Therefore, novel and effec+ve an+bio+cs are essen+al for the con+nued treatment of bacterial infec+ons. A targetspecific an+ bacterial assay was used to iden+fy compounds from A. californica that inhibited bacterial growth by inhibi+ng the FabI enzyme. Compounds which demonstrate decreased potency against a bacterial strain overexpressing FabI compared to a control strain have been isolated and characterized. The decreased ac+vity in the overexpressing FabI strain suggests that the mode of ac+on of this flavonoid is FabI inhibi+on. Introduc.on For thousands of years, natural products have been used as medicinal purposes. Therefore they are great candidates for poten+al source of new drugs. 1 Artemisia californica has been used for medicinal purposes such as treatment of cuts, wounds, infec+on and coughs by the Chumash Na+ve Americans. 2 Previous work in our lab has shown that a flavonoid compound from costal sage extract has an+bacterial ac+vity. Unfortunately, not enough of the compound has been isolated to iden+fy its chemical structure and proper+es. The objec+ve of this project is to isolate the flavonoid compound from A. californica so that its structure and biological ac+vity can be fully characterized. Experimental Methods The ini+al A. californica sample used in this project (Frac+on 1) was taken from the results of a previous project in our lab. Chromatographic separa+ons were performed using a reverse phase HPLC system using a C 18 column with a methanolwater gradient as the mobile phase. Frac+on 1 was frac+onated using a prepara+ve scale column with a 55%100% MeOH gradient over 60 minutes and the eluent was collected into separate vials using a frac+on collector. The collected eluent was separated into five frac+ons and the solvent was removed by using a rotary evaporator. The third frac+on (1C) contained the compound of interest and was further separated using a semiprepara+ve scale with a 60%66% MeOH gradient over 30 minutes. The eluent was again collected using a frac+on collector and separated into four frac+ons and the solvent was removed using a rotary evaporator. The third frac+on (1C2) was found to contain the compound of interest and was further frac+onated using a semiprepara+ve scale column with a 60%64% MeOH gradient over 30 minutes. Conclusions • The peaks in the mass spectra with m/z values of 301, 316 and 331 indicate that the compound of interest was isolated from the parent frac+ons in each frac+ona+on step. • Not enough of the compound of interest was purified to allow for complete structural characteriza+on of the compound using NMR. Future Direc.ons • Future efforts will a\empt isolate a greater amount of the compound of interest so that its structure can be fully determined. • Once the structure of the an+bacterial compound is characterized, further work will seek to more fully characterize its biological proper+es. Acknowledgements This project was funded by an Undergraduate Research Fellowship through the Natural Science Division of Pepperdine University. We would also like to thank Bri\any Allison for providing A. californica extracts used in this study. References (1) Butler, M. S. Journal of Natural Products 2004, 67, 21412153. (2) Timbrook, J. Chumash Ethnobotany: Plant Knowledge Among the Chumash People of Southern California; Heyday Books, 2007. (3) Dale, N. Flowering Plants: The Santa Monica Mountains, Coastal and Chaparral Regions of Southern California; 1st ed.; Capra Press, 1986. Results Frac.on 1 Frac.on 1A Frac.on 1B Frac.on 1D Frac.on 1C Frac.on 1E Frac.on 1C1 Frac.on 1C2 Frac.on 1C3 Frac.on1 C4 Frac.on 1C2a Frac.on 1C2b Frac.on 1C2c Frac.on 1C2d Frac.on 1C2b Flow Chart of Frac.ona.on Chromatograms of Ac.ve Frac.ons Mass Spectrum of Boxed Region in Each Chromatogram Figure 1. The presence of the compound of interest in this project was confirmed in Frac+on 1 using LCMS analysis. A series of chromatographic separa+ons were then performed which generated the frac+ons shown above. Frac+ons containing the compound of interest with an+bacterial ac+vity (light blue) were selected at each stage for further frac+ona+on. Each frac+ona+on was performed using HPLC with a C18 column as described in the experimental sec+on. Figure 2. The chromatograms corresponding to each of the frac+ons with an+bacterial ac+vity from Figure 1. A red box is shown around the region selected for further frac+ona+on in each chromatogram. Figure 3. The mass spectrum of the boxed region in each of the chromatograms shown in Figure 2. Each spectrum contains peaks with m/z values of 301, 316 and 331, which correspond to compound of interest. An.bacterial Frac.on An.bacterial Frac.on RT: 0.00 - 60.00 0 5 10 15 20 25 30 35 40 45 50 55 Time (min) 0 50000 100000 150000 200000 250000 300000 350000 400000 450000 500000 550000 600000 650000 700000 750000 uAU NL: 7.89E5 Total Scan PDA JM060513 A_50mg_o n_JM05281 3A_2 Region selected for further frac+ona+on RT: 0.00 - 30.00 SM: 7B 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (min) 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 uAU NL: 1.91E5 Total Scan PDA JM062413A _0_2964mg_ of_JM06051 Region selected for further frac+ona+on RT: 0.00 - 30.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (min) 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 220000 240000 260000 280000 300000 320000 uAU NL: 3.30E5 Total Scan PDA JM062813A _2_91mg_o f_JM06241 Region selected for further frac+ona+on JM060513A_50mg_on_JM052813A_2 # 1535-1702 RT: 39.96-44.92 AV: 168 NL: 8.06E2 T: + p ESI sid=5.00 Full ms [80.00-1200.00] 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 Intensity 301.20 316.27 331.27 JM062413A_0_2964mg_of_JM060513C2_4 # 446-514 RT: 13.34-15.38 AV: 69 NL: 2.98E3 T: + p ESI sid=5.00 Full ms [80.00-1200.00] 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 Intensity 301.20 331.27 316.27 JM062813A_2_91mg_of_JM062413B_3 # 451-525 RT: 13.09-15.25 AV: 75 SM: 7B NL: 5.05E3 T: + p ESI sid=5.00 Full ms [80.00-1200.00] 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 Intensity 316.27 301.20 331.27 0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 4 5 6 7 8 9 10 OD @ 600 nm Time (hours) Negative control Flavonoid treatment (120 nM) Bacteria grow normally when treated with only the solvent vehicle (negative contol). Bacterial growth is significantly reduced when treated with flavonoid (120 nM) from A. californica. Isola+on of An+bacterial Compounds from Artemisia californica Jae Eun Min and P. Ma\hew Joyner Department of Chemistry, Natural Science Division, Pepperdine University, Malibu, California, USA
Transcript of Isolation of Antibacterial Compounds from Artemisia ...
Abstract The Chumash Na+ve Americans of Southern California have well-‐documented tradi+ons of using plants for medicinal purposes. If a specific plant has tradi+onally been used by Chumash for the treatment of cuts, wounds and infec+ons, it may contain chemicals with an+-‐bacterial proper+es. One plant that fits these criteria is Artemisia californica (coastal sage). Because of the widespread use of an+bio+cs over the past sixty years bacteria are evolving greater resistance to known an+bio+cs, but unfortunately the rate of an+bio+c discovery has diminished during the past twenty years. Therefore, novel and effec+ve an+bio+cs are essen+al for the con+nued treatment of bacterial infec+ons. A target-‐specific an+-‐bacterial assay was used to iden+fy compounds from A. californica that inhibited bacterial growth by inhibi+ng the FabI enzyme. Compounds which demonstrate decreased potency against a bacterial strain over-‐expressing FabI compared to a control strain have been isolated and characterized. The decreased ac+vity in the over-‐expressing FabI strain suggests that the mode of ac+on of this flavonoid is FabI inhibi+on.
Introduc.on For thousands of years, natural products have been used as medicinal purposes. Therefore they are great candidates for poten+al source of new drugs.1 Artemisia californica has been used for medicinal purposes such as treatment of cuts, wounds, infec+on and coughs by the Chumash Na+ve Americans.2 Previous work in our lab has shown that a flavonoid compound from costal sage extract has an+bacterial ac+vity. Unfortunately, not enough of the compound has been isolated to iden+fy its chemical structure and proper+es. The objec+ve of this project is to isolate the flavonoid compound from A. californica so that its structure and biological ac+vity can be fully characterized.
Experimental Methods The ini+al A. californica sample used in this project (Frac+on 1) was taken from the results of a previous project in our lab. Chromatographic separa+ons were performed using a reverse phase HPLC system using a C18 column with a methanol-‐water gradient as the mobile phase. Frac+on 1 was frac+onated using a prepara+ve scale column with a 55%-‐100% MeOH gradient over 60 minutes and the eluent was collected into separate vials using a frac+on collector. The collected eluent was separated into five frac+ons and the solvent was removed by using a rotary evaporator. The third frac+on (1C) contained the compound of interest and was further separated using a semiprepara+ve scale with a 60%-‐66% MeOH gradient over 30 minutes. The eluent was again collected using a frac+on collector and separated into four frac+ons and the solvent was removed using a rotary evaporator. The third frac+on (1C2) was found to contain the compound of interest and was further frac+onated using a semiprepara+ve scale column with a 60%-‐64% MeOH gradient over 30 minutes.
Conclusions • The peaks in the mass spectra with m/z values of 301, 316 and
331 indicate that the compound of interest was isolated from the parent frac+ons in each frac+ona+on step.
• Not enough of the compound of interest was purified to allow for complete structural characteriza+on of the compound using NMR.
Future Direc.ons • Future efforts will a\empt isolate a greater amount of the
compound of interest so that its structure can be fully determined.
• Once the structure of the an+bacterial compound is characterized, further work will seek to more fully characterize its biological proper+es.
Acknowledgements T h i s p r o j e c t w a s f u n d e d b y a n Undergraduate Research Fellowship through the Natural Science Division of Pepperdine University. We would also like to thank Bri\any Allison for providing A. californica extracts used in this study.
References (1) Butler, M. S. Journal of Natural Products 2004, 67, 2141-‐2153. (2) Timbrook, J. Chumash Ethnobotany: Plant Knowledge Among the
Chumash People of Southern California; Heyday Books, 2007. (3) Dale, N. Flowering Plants: The Santa Monica Mountains, Coastal and
Chaparral Regions of Southern California; 1st ed.; Capra Press, 1986.
Results
Frac.on 1
Frac.on 1A Frac.on 1B Frac.on 1D Frac.on 1C Frac.on 1E
Frac.on 1C1
Frac.on 1C2
Frac.on 1C3
Frac.on1C4
Frac.on 1C2a
Frac.on 1C2b
Frac.on 1C2c
Frac.on 1C2d
Frac.on 1C2b
Flow Chart of Frac.ona.on Chromatograms of Ac.ve Frac.ons Mass Spectrum of Boxed Region in Each Chromatogram
Figure 1. The presence of the compound of interest in this project was confirmed in Frac+on 1 using LCMS analysis. A series of chromatographic separa+ons were then performed which generated the frac+ons shown above. Frac+ons containing the compound of interest with an+bacterial ac+vity (light blue) were selected at each stage for further frac+ona+on. Each frac+ona+on was performed using HPLC with a C18 column as described in the experimental sec+on.
Figure 2. The chromatograms corresponding to each of the frac+ons with an+bacterial ac+vity from Figure 1. A red box is shown around the region selected for further frac+ona+on in each chromatogram.
Figure 3. The mass spectrum of the b o x e d r e g i o n i n e a c h o f t h e chromatograms shown in Figure 2. Each spectrum contains peaks with m/z values of 301, 316 and 331, which correspond to compound of interest.
An.bacterial Frac.on
An.bacterial Frac.on
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JM060513A_50mg_on_JM052813A_2 #1535-1702 RT: 39.96-44.92 AV: 168 NL: 8.06E2T: + p ESI sid=5.00 Full ms [80.00-1200.00]
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JM062413A_0_2964mg_of_JM060513C2_4 #446-514 RT: 13.34-15.38 AV: 69 NL: 2.98E3T: + p ESI sid=5.00 Full ms [80.00-1200.00]
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Negative control Flavonoid treatment (120 nM)
Bacteria grow normally when treated with only the solvent vehicle (negative contol).
Bacterial growth is significantly reduced when treated with flavonoid (120 nM) from A. californica.
Isola+on of An+bacterial Compounds from Artemisia californica Jae Eun Min and P. Ma\hew Joyner
Department of Chemistry, Natural Science Division, Pepperdine University, Malibu, California, USA
