Curcumin Research - Revision history

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2019-09-05T15:34:55Z

Literature references

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2019-09-05T15:33:15Z

Literature references

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2019-09-05T15:30:38Z

Literature references

Ssaey at 00:27, 11 May 2018

2018-05-11T00:27:54Z

Ssaey at 00:18, 11 May 2018

2018-05-11T00:18:55Z

Ssaey at 00:09, 11 May 2018

2018-05-11T00:09:37Z

Ssaey at 00:06, 11 May 2018

2018-05-11T00:06:48Z

Ssaey at 23:57, 10 May 2018

2018-05-10T23:57:05Z

Ssaey at 23:29, 10 May 2018

2018-05-10T23:29:32Z

Ssaey at 23:27, 10 May 2018

2018-05-10T23:27:54Z

@@ Line 90: / Line 90: @@
 Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). [[Media:1-s2.0-S1570023213004327-main.pdf|Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy.]] ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937,  doi:10.1016/j.jchromb.2013.08.011
-Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.
+Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. [[Media:41_1640.pdf|Nematocidal activity of turmeric: synergistic action of curcuminoids.]] (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.
 ===10. Signature===
 Two copies of the report will be signed and dated and turned in to the Faculty Research Advisor and archived by the Research Coordinator.

@@ Line 88: / Line 88: @@
 Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). [[Media:1-s2.0-S0006295207005758-main.pdf|Curcumin as ‘curecumin’: from kitchen to clinic.]]  ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016
-Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy. ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937,  doi:10.1016/j.jchromb.2013.08.011
+Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). [[Media:1-s2.0-S1570023213004327-main.pdf|Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy.]] ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937,  doi:10.1016/j.jchromb.2013.08.011
 Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.

@@ Line 86: / Line 86: @@
 ===Literature references===
-Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). Curcumin as ‘curecumin’: from kitchen to clinic.  ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016
+Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). [[Media:1-s2.0-S0006295207005758-main.pdf|Curcumin as ‘curecumin’: from kitchen to clinic.]]  ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016
 Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy. ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937,  doi:10.1016/j.jchromb.2013.08.011

@@ Line 14: / Line 14: @@
 ==Written Report==
-===1. Descriptive information===
+===Descriptive information===
 '''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''
@@ Line 23: / Line 23: @@
 Research work documented in lab notebooks.
-===2. Background from earlier reports===
+===Background from earlier reports===
 Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.
-===3. Experimental===
+===Experimental===
 :'''Sourcing turmeric:'''
 In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company.  We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].
@@ Line 79: / Line 79: @@
 :'''Nuclear Magnetic Resonance (NMR) Testing:'''
 ::Each fraction was rotovapped so that all solvent was removed, and then About 1mg of each curcumin product was added to separate NMR tubes. 1mL of duderated DMSO was added to resuspend the curcumin samples to be used in the 400Hz NMR at Knox College. Only fraction 1 (presumed to be bisdemethoxycurcumin) was analyzed at the time.
-[[File:fraction 1.png|400px|thmb|none|Figure 3: NMR spectrum for the first fraction collected during Flash Chromatography. Notable peaks include those in the aromatic region, as well as the methoxy region. The large peak at 4.5 is DMSO.]]
+[[File:fraction 1.png|500px|thumb|none|Figure 3: NMR spectrum for the first fraction collected during Flash Chromatography. Notable peaks include those in the aromatic region, as well as the methoxy region. The large peak at 4.5 is DMSO.]]
-===6. Discussion===
+===Discussion===
-The flash chromatography and HPLC data have been consistent with the literature regarding the existence of curcumin's three different molecular structures. However, characterization of curcumins through NMR have led to further questions on  the structures present within each curcumin sample. Further interpretation and testing are been necessary to have certainty in the purity of our samples as well as specific characterization of each fraction.
+The flash chromatography and HPLC data have been consistent with the literature regarding the existence of curcumin's three different molecular structures. However, characterization of curcuminoids through NMR have led to further questions on  the structures present within each curcumin sample. The first fraction eluted through Flash Chromatography using the method described above should be bisdemthoxycurcumin, as this derivative is the most polar. However, the literature agrees that the most nonpolar derivative (curcumin I) is the most abundant within the turmeric plant and therefore it remains unclear why the first peak yields the greatest absorbance and product if it is not curcumin I. It is possible that the first large peak on the Flash Chromatogram exists as a complex of all three derivates, and that the second and third peaks are unknown compounds such as the turmeric oleoresins. Further interpretation and testing are been necessary to have certainty in the purity of our samples as well as specific characterization of each fraction. The general consensus seems to be that the three derivatives are simply too similar in structure to be separated with the proposed method.
-===7. Conclusions===
+===Literature references===
 Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). Curcumin as ‘curecumin’: from kitchen to clinic.  ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016

@@ Line 55: / Line 55: @@
 :'''Thin Layer Chromatography'''
-::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Figure A. Final Rf values were recorded.
+::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Image C. Final Rf values were recorded.
-[[File:Example.jpg|200px|thumb|none|Figure A: TLC results yielded three distinct and visible lines  for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.]]
+[[File:Example.jpg|200px|thumb|none|Image C: TLC results yielded three distinct and visible lines  for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.]]
 :'''Concentration of Crude Extract'''
@@ Line 62: / Line 62: @@
 ::The cooler was set to 8.1 degrees Celcius and the water bath was set to 45 degrees Celcius. The speed was set at 5.
 ::Approximately 80 mL of the crude curcumin mixture was pipetted into a 500 mL rbf. The solvent was removed via the rotovap instrument for about 80 minutes. The resulting concentrated crude extract was transferred to a 20 mL glass vile and allowed to separate into a bottom, red layer (containing the curcuminoids) and a top, yellow layer (containing the oleoresin). The oleoresin was removed from the curcuminoids via a pipette and discarded.
-[[File:rotovapped crude mixture.png|200px|Image C: Crude extract following rotovapping; bottom layer (dark red) contains curcuminoids, top layer (yellow) is oleoresin]]
+[[File:rotovapped crude mixture.png|200px|thumb|none|Image C: Crude extract following rotovapping; bottom layer (dark red) contains curcuminoids, top layer (yellow) is oleoresin]]
 :'''Flash Column Chromatography:'''
 ::An 100g gold C18 reverse phase Column was used to run 2mL of the concentrated curcuminoid extract.  This column uses acetyl nitrile and acidic(TFA 0.1%) water to run the liquid phase through the column.
-:: A gradient was ran for 90 min (start: 10% ACN/100% H2O, finish: 100% ACN) that yielded three distinct peaks.**  The fractions for each peak were collected and stored in separate flasks.
+:: A gradient was ran for 90 min (start: 10% ACN/100% H2O, finish: 100% ACN) that yielded three distinct peaks. The fractions for each peak were collected and stored in separate flasks.
-**Method is saved as "ssaeycurcuminsummer17" in the Flash Chromatography instrument.
+Method is saved as "ssaeycurcuminsummer17" in the Flash Chromatography instrument.
 :'''High Pressure Liquid Chromatography (HPLC):'''
 ::Each 1mL of each Flash eluent was added to a separate HPLC vile and labeled to be compared to the original Curcumin solution extracted through Soxhlet extraction.
-[[File:T2 Curcumin HPLC chromatogram.jpg|500px|thumb|none|HPLC chromatogram]]
+[[File:T2 Curcumin HPLC chromatogram.jpg|500px|thumb|none|Figure 2: HPLC chromatogram]]
 :'''Rotovaporization''':
 :: Each fraction of curcumin had its solvent extracted via rotovaportization. The hot bathwater was held at 45 degrees Celsius for about an hour and a half or until completely dry. Spin speed was between 9 and 10, and adjustments  in the angle of inundation of the round bottom flask containing the curcumin fractions was adjusted as the solvent was vaporized.
 :'''Nuclear Magnetic Resonance (NMR) Testing:'''
-::Each fraction was rotovapped so that all solvent was removed, and then About 1mg of each curcumin was added to separate NMR tubes. 1mL of duderated DMSO was added to resuspend the curcumin samples to be used in the 400Hz NMR at Knox College.
+::Each fraction was rotovapped so that all solvent was removed, and then About 1mg of each curcumin product was added to separate NMR tubes. 1mL of duderated DMSO was added to resuspend the curcumin samples to be used in the 400Hz NMR at Knox College. Only fraction 1 (presumed to be bisdemethoxycurcumin) was analyzed at the time.
+[[File:fraction 1.png|400px|thmb|none|Figure 3: NMR spectrum for the first fraction collected during Flash Chromatography. Notable peaks include those in the aromatic region, as well as the methoxy region. The large peak at 4.5 is DMSO.]]
 ===6. Discussion===

@@ Line 30: / Line 30: @@
 In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company.  We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].
-''Procedure for extraction was modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.
+''Procedure for extraction was modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull 1993;41:1640-3.
 :[[Media:Turmeric ref 01.pdf|Reference]]
@@ Line 61: / Line 61: @@
 ::The cooler was set to 8.1 degrees Celcius and the water bath was set to 45 degrees Celcius. The speed was set at 5.
 ::Approximately 80 mL of the crude curcumin mixture was pipetted into a 500 mL rbf. The solvent was removed via the rotovap instrument for about 80 minutes. The resulting concentrated crude extract was transferred to a 20 mL glass vile and allowed to separate into a bottom, red layer (containing the curcuminoids) and a top, yellow layer (containing the oleoresin). The oleoresin was removed from the curcuminoids via a pipette and discarded.
-[[File:rotovapped crude mixture.png|400px]]
+[[File:rotovapped crude mixture.png|200px|Image C: Crude extract following rotovapping; bottom layer (dark red) contains curcuminoids, top layer (yellow) is oleoresin]]
 :'''Flash Column Chromatography:'''
@@ Line 73: / Line 73: @@
 :'''Rotovaporization''':
-:: Each fraction of curcumin had its solvent extracted via rotovaportization. The hot bathwater was held at 45 degrees Celsius for about an hour and a half or until completely dry. Spin speed was between 9 and 10, and adjustments  in the angle of inundation of the round bottom flask containing the curcumin fractions was adjusted as the solvent was vaporized.[[File:flashdata.png|400px]]
+:: Each fraction of curcumin had its solvent extracted via rotovaportization. The hot bathwater was held at 45 degrees Celsius for about an hour and a half or until completely dry. Spin speed was between 9 and 10, and adjustments  in the angle of inundation of the round bottom flask containing the curcumin fractions was adjusted as the solvent was vaporized.
 :'''Nuclear Magnetic Resonance (NMR) Testing:'''
-::About 1mg of each curcumin was added to separate NMR tubes. 1mL of duderated DMSO was added to resuspend the curcumin samples to be used in the 400Hz NMR at Knox College.
+::Each fraction was rotovapped so that all solvent was removed, and then About 1mg of each curcumin was added to separate NMR tubes. 1mL of duderated DMSO was added to resuspend the curcumin samples to be used in the 400Hz NMR at Knox College.
 ::'''TLC Results'''
 :TLC results from two trials of the diluted EtOAc/curcuminoid mixture (prepared from MeOH in soxhlet extraction) are shown in Figure A. A RediSep silica TLC plate was used with an 8:1 chloroform:acetone solvent front. Three distinct lines are visible for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.
 [[File:Example.jpg|200px|thumb|none|Figure A: TLC Results]]
 ::'''HPLC Results'''

@@ Line 23: / Line 23: @@
 Research work documented in lab notebooks.
+===2. Background from earlier reports===
 Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.
-===4. Experimental===
+===3. Experimental===
 :'''Sourcing turmeric:'''
 In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company.  We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].
@@ Line 36: / Line 35: @@
 :'''Methanol in Soxhlet Extraction:'''
 ::1. Soxhlet apparatus was set up for extraction, as pictured in Image A.
-::2. Approximately 76 grams of organic turmeric from Starwest Botanicals was added to the thimble.
+::2. Approximately 80 grams of organic turmeric from Starwest Botanicals was added to the thimble.
-::3. Approximately 350 mL of MeOH was added to the stillpot and a few boiling stones were added.
+::3. Approximately 300 mL of MeOH was added to the stillpot and a few boiling stones were added.
 ::4. Heat source and water source were turned on.
 ::5. Extraction was ran for 6 hours.
@@ Line 49: / Line 48: @@
 ::4. The stopcock was removed, allowing the mixture to separate into two separate phases, one containing EtOAc and curcuminoids, and the other containing MeOH, water, and impurities. Image B displays this separated mixture.
 ::5. The EtOAc/curcuminioid phase (top layer) was drained into a labeled glassware container.
-::6.The bottom layer was readded to the separatory funnel along with 150 mL of EtAOc, 150 mL water, and 100 mL brime.
+::6.The bottom layer was readded to the separatory funnel along with 150 mL of EtOAc, 150 mL water, and 100 mL brine.
 ::7. Again, the stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
 ::8. The stopcock was removed and the mixture was allowed to separate.
-::9. The top layer was added to the labeled glassware and the bottom layer was discarded.
+::9. The top layer was added to the labeled glassware and the bottom layer was discarded. The entire process resulted in approximately 250 mL of crude curcumin mixed with the solvents used for extraction.
 [[File:separatoryfunnel.jpg|200px|thumb|left|Image B: Separation of EtOAc and curcuminoids (top layer) from MeOH and impurities (bottom layer)]]
 :'''Thin Layer Chromatography'''
 ::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Figure A. Final Rf values were recorded.
 :'''Flash Column Chromatography:'''
-::A 15.5mL(13g) gold C18 reverse phase Column was used to run 1mL of the EtOAc/curcuminoid extract.  This column uses acetyl nitrile and acidic(TFA 0.1%) water to run the liquid phase through the column.
+::An 100g gold C18 reverse phase Column was used to run 2mL of the concentrated curcuminoid extract.  This column uses acetyl nitrile and acidic(TFA 0.1%) water to run the liquid phase through the column.
-:: A gradient was ran for 25 min that yielded three distinct peaks.  The eluents for each peak were collected and stored in separate viles.
+:: A gradient was ran for 90 min (start: 10% ACN/100% H2O, finish: 100% ACN) that yielded three distinct peaks.**  The fractions for each peak were collected and stored in separate flasks.
 :'''High Pressure Liquid Chromatography (HPLC):'''
@@ Line 67: / Line 73: @@
 :'''Rotovaporization''':
-:: Each fraction of curcumin had its solvent extracted via rotovaportization. The hot bathwater was held at 32 degrees Celsius for about an hour and a half or until completely dry. Spin speed was between 9 and 10, and adjustments  in the angle of inundation of the round bottom flask containing the curcumin fractions was adjusted as the solvent was vaporized.
+:: Each fraction of curcumin had its solvent extracted via rotovaportization. The hot bathwater was held at 45 degrees Celsius for about an hour and a half or until completely dry. Spin speed was between 9 and 10, and adjustments  in the angle of inundation of the round bottom flask containing the curcumin fractions was adjusted as the solvent was vaporized.[[File:flashdata.png|400px]]
 :'''Nuclear Magnetic Resonance (NMR) Testing:'''

@@ Line 8: / Line 8: @@
 Curcumin I (Main Curcuminoid, in picture below), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the three major curcuminoids present in turmeric and of interest to this study.  Their respective chemical structures are depicted below:
-[[Media:Curcuminoid Analogs with Potent Activity.pdf|Curcuminoid Analogs with Potent Activity (Article)]]
+Structures retrieved from: [[Media:Curcuminoid Analogs with Potent Activity.pdf|Curcuminoid Analogs with Potent Activity (Article)]]
 [[File:Curcumin.jpg|400px|thumb|none|Curcumin I, II, and III]]

← Older revision		Revision as of 23:27, 10 May 2018
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	''Procedure for extraction was modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.		''Procedure for extraction was modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.
		+	:[[Media:Turmeric ref 01.pdf\|Reference]]

	:'''Methanol in Soxhlet Extraction:'''		:'''Methanol in Soxhlet Extraction:'''