extraction of natural products from the brown alga dictyota · ms ms/ms . references abou-el-wafa,...
TRANSCRIPT
Extraction of Natural Products from the Brown Alga Dictyota Ashley Nicoll, Megan Rooney, Dr. Evgenia Glukhov, and Dr. William Gerwick
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093, United States
From the sample of Dictyota, 10 fractions were collected. The fractions contained many different unknown compounds that could be seen using TLC (thin layer chromatography) and LCMS. The unknown compounds in the fractions could be further elucidated and investigated in the future.
VACUUM LIQUID CHROMATOGRAPHY
EXTRACTING ALGAE
1. A Buchner funnel is used to filter the algae, wrapped in a cheese clothe, after soaking it in DCM:MeOH solution for 45 minutes.
2. A vacuum is applied to the vacuum flask so that the liquid will run through the funnel and into the flask.
3. The extract that has run through the funnel is collected in the flask. Next, it will be put on a RotoVap to evaporate off the DCM and MeOH and just leave the extract from the algae.
Thank you to Nathan Moss and Bailey Miller for collecting the Dictyota sample used and to everyone in the lab for their teaching.
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The RotoVap evaporates off volatile organic solvents from solutions to leave just the organic compounds.
The column is made of tightly packed silica so that there are no air bubbles in the column.
The sample is dissolved in the first solvent, the least polar, and added to the top. Vacuum is then applied and the non-polar parts of the sample move through the column to be collected in the bottom flask as fraction A.
As different solvents are added, the more polar compounds begin to move down the column in bands.
At the end, methanol is run through the column until there are no bands left in the column.
ABSTRACT
THE ROTOVAP
LCMS PROFILE AND MOLECULAR NETWORKING
CONCLUSION
ACKNOWLEDGEMENTS
A B C D E F G H Ia Ib A B C D E F G H Ia Ib
Biologically active secondary metabolites have become a new focus in the search
for natural product drugs. These metabolites can be isolated from a number of different
marine and terrestrial resources. The Gerwick lab works on analyzing marine entities for
biologically active secondary metabolites. Many projects begin with algae that have
been collected from different parts of the world. In this study, the alga Dictyota was
collected from American Samoa on July, 16, 2014. Dictyota is a genus of brown algae;
the main compounds isolated from Dictyota in the past have been diterpenes and other
non-polar compounds. Recently, several new diterpenes have been isolated from
different species of Dictyota (Ghada S. E. Abou-El-Wafa et al., 2013).
The process of finding out what compounds could be in the sample of Dictyota
began with using DCM:MeOH solution to extract the biomass. Next a VLC (vacuum
liquid chromatography) column was used to separate the extract into fractions based on
polarity. The solvents used were hexanes, ethyl acetate, and methanol in varying
proportions. A small portion of each fraction was then run through the LCMS (liquid
chromatography mass spectrometry). Next, the data from the LCMS was used in GNPS
(Global Natural Product Social) to perform molecular networking. Molecular networking
shows what compounds could be in the sample and which compounds are similar to
one another. Molecular networking coupled with raw data from LCMS can be used to
predict which compounds could be in a sample. If a new compound is found, more tests
can be done to further elucidate its structure and test its properties.
Above: A picture of the MS and MS/MS data for a peak occurring at 25.42 minutes.
Below: A picture of the cluster where the compound that the peak represents is found. The compound appears in green.
C:\Users\...\OTHERS\2231\7-21-15_2231Ia 7/21/2015 8:19:09 PM
RT: 0.00 - 29.96
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29Time (min)
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Rel
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11.12 25.42
20.48
18.67 25.8919.3022.22
2.32 14.74 23.2421.6415.102.521.38 25.2024.1717.586.013.06 7.40 17.423.71 4.41 9.790.49 16.258.63 13.8011.865.19 10.267.676.21 9.72 12.39 27.1726.45 28.20
NL:7.02E6Base Peak F: + c ESI Full ms [190.00-2000.00] MS 7-21-15_2231Ia
7-21-15_2231Ia #963-977 RT: 25.31-25.52 AV: 3 NL: 3.13E6F: + c ESI Full ms [190.00-2000.00]
760 765 770 775 780 785 790 795 800 805 810 815 820 825 830 835 840m/z
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Rel
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793.87
794.83
789.46
788.63790.28 795.76
816.60796.67767.96 791.83 812.55 839.78803.74764.91 787.83 825.86799.86 821.67808.88 818.54769.61762.01 776.87 823.96774.46 834.04784.10772.01 780.69 806.56 831.80 836.97829.56
7-21-15_2231Ia #963-977 RT: 25.45-25.55 AV: 2 NL: 1.47E6F: + c ESI d Full ms2 [email protected] [205.00-805.00]
220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800m/z
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Rel
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517.36
794.39
518.27
793.48 795.37491.83 543.28 631.31256.46 648.10 685.58301.07 341.14280.75 774.48233.42 745.42716.59662.16461.72441.85377.36316.32 708.27426.48 523.26
MS
MS/MS
REFERENCES
Abou-El-Wafa, Ghada S. E., Mohamed Shaaban, Khaled A. Shaaban, Mohamed E. E. El-Naggar, Armin Maier, Heinz H. Fiebig, and Hartmut Laatsch. "Pachydictyols B and C: New Diterpenes from Dictyota dichotoma Hudson." Marine Drugs. MDPI, 22 Aug. 2013.
The extraction process was repeated 4 times, until the extract color was very light.
American Samoa Dictyota
Interest