Difference between revisions of "Curcumin Research"

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<!-- ==Curcumin Research Initiative== -->
 
<!-- ==Curcumin Research Initiative== -->
<!-- ==The Three Curcuminoids== -->
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You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project.  This page was created by [[Stehanie_Saey|Stephanie Saey]] and Nadia Ayala. Nadia was a 2017 Biochemistry graduate and Stephanie was a 2018 Biochemistry/Biopsychology graduate.
<!-- ==Flash Chromatography Data== -->
 
<!-- ==NMR Data== -->
 
<!-- ==Analyses and Discussion== -->
 
<!-- ==Looking Ahead== -->
 
<!-- -->
 
<!-- -->
 
  
You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project.  This page was created and is maintained by Stephanie Saey and Nadia Ayala.
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===Abstract ===
 
+
Turmeric, Curcuma longa, is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties. The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography. However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids (I/II/III) in amounts large enough for further investigation on its radicalisation properties.
==Curcumin Research Initiative==
 
 
 
===Preparation of Curcuminoid Standards from Turmeric Plant===
 
Curcumin is a secondary plant metabolite of the turmeric herb ''Curcuma longa''. The term "curcumin" has been used to refer to the bioactive molecule, but in reality curcumin has three derivatives (I/II/III) of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Together, the aforementioned compounds are known as curcuminoids. A review of literature suggests that curcuminoids have chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered.  However, difficulty arises when seeking to study these curcuminoids individually. Curcumin I is commercially available (10 mg for $150), while demethoxycurcumin (II) and bisdemethoxycurcumin (III) are not commercially available. Our current research project aims to isolate (methanol in soxhlet), purify (automated Flash/HPLC), and characterize (NMR) the curcuminoids for further investigation.
 
  
 
===The Three Curcuminoids===
 
===The Three Curcuminoids===
 
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:
 
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]]
 
[[File:Curcumin.jpg|400px|thumb|none|Curcumin I, II, and III]]
 
===Flash Chromatography Data===
 
 
We used a 15.5mL C18 Column to run 1mL of our Curcumin extract. This column uses acetyl nitrile and water to run the liquid phase through the column. We ran a gradient for 25 min and got three distinct peaks which we then proceeded to run an HPLC method on.
 
 
===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].
 
 
===Overview of Proposed Methodology===
 
''Procedure is 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.
 
 
*Extract curcuminoids with MeOH under soxhlet extraction.
 
*Partition MeOH supernatant with ethyl acetate (AcOEt) and water for purification purposes.
 
*Fractionate AcOEt extract by silica gel column chromatography to yield AcOEt curcumin eluents.
 
*Fractionate AcOEt eluents by silica gel TLC using chloroform: acetone = 8:1 as the solvent system.
 
*Purify each eluent by removing organic solvent (?) and analyzing under HPLC.
 
 
===Extracting Curcuminoids under Soxhlet Distillation===
 
*Weigh out 150.00g of turmeric powder with an analytical balance.
 
*Measure out 300.00mL MeOH using a burette.
 
*Place above compound and solvent in stillpot of soxhlet apparatus.
 
*Carefully turn on heat source and water source.
 
*Run extraction for 6-7 hours.
 
  
 
==Written Report==
 
==Written Report==
  
===1. Descriptive information===
+
===Descriptive information===
 
'''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''
 
'''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''
  
Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon#
+
Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon
  
 
Special thanks to Michael Prinsell and Broddie Davis  
 
Special thanks to Michael Prinsell and Broddie Davis  
  
Research work documented in lab notebooks, Stephanie Saey pages 1-13 and Nadia Ayala pages [insert NA page#].
+
Research work documented in lab notebooks.  
 
 
===2. Introduction===
 
  
Turmeric, ''Curcuma longa,'' is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties (Goel, 2008). The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives (I/II/III) of different molecular structures: curcumin I, demethoxycurcumin (II), and bisdemethoxycurcumin (III).  Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography (Jayaprakasha).  However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually.  Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids in amounts large enough for further investigation.
+
===Background from earlier reports===
 
 
===3. 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.  
 
Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.  
  
===4. Experimental===
+
===Experimental===
 
:'''Sourcing turmeric:'''
 
:'''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].
 
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 1993;41:1640-3.
 +
:[[Media:Turmeric ref 01.pdf|Reference]]
  
 
:'''Methanol in Soxhlet Extraction:'''
 
:'''Methanol in Soxhlet Extraction:'''
 
::1. Soxhlet apparatus was set up for extraction, as pictured in Image A.  
 
::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.  
 
::4. Heat source and water source were turned on.  
 
::5. Extraction was ran for 6 hours.  
 
::5. Extraction was ran for 6 hours.  
Line 82: 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.  
 
::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.  
 
::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.
 
::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.  
 
::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)]]
 
[[File:separatoryfunnel.jpg|200px|thumb|left|Image B: Separation of EtOAc and curcuminoids (top layer) from MeOH and impurities (bottom layer)]]
  
 
:'''Thin Layer Chromatography'''
 
:'''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|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'''
 +
::Used rotovap (in Organic Chemistry lab) to concentrate the curcumin mixture before running Flash Chromatography.
 +
::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|thumb|none|Image C: Crude extract following rotovapping; bottom layer (dark red) contains curcuminoids, top layer (yellow) is oleoresin]]
  
 
:'''Flash Column Chromatography:'''
 
:'''Flash Column Chromatography:'''
::A 15.5mL(13g) C18 Column was used to run 1mL of the EtOAc/curcuminoid extract.  This column uses acetyl nitrile and 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.
 +
Method is saved as "ssaeycurcuminsummer17" in the Flash Chromatography instrument.
 +
[[File:flashdata.png|500px|thumb|none|Figure 1: Flash Chromatography separation of the crude curcumin extract. The first peak was presumed to be bidemethoxycurcumin (curcumin III), the most polar of the three curcuminoids, followed by demethoxycurcumin (curcumin II) and curcumin I (most nonpolar).]]
  
 
:'''High Pressure Liquid Chromatography (HPLC):'''
 
:'''High Pressure Liquid Chromatography (HPLC):'''
::Each Flash eluent was added to a separate HPLC vile.  Additionally, a "standard" was made by diluting 1 mL of the original EtOAc/curcuminoid mixture in 4 mL EtOAc. 1mL of the resulting dilution was placed into its on HPLC vile. The HPLC was run for 20 minutes under bes method .... [methods for this portion to be continued when I am able to refer back to the HPLC data...the HPLC is currently under repair].
+
::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|Figure 2: HPLC chromatogram]]
:'''Preparation for Nuclear Magnetic Resonance (NMR) Testing:'''
 
::The flash chromatography eluents were each added to their own 50 mL rbf and separately rotovapped for approximately 1 hour and 20 min to remove solvent.
 
 
 
===5. Results===
 
::'''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]]
 
 
 
::'''Flash Chromatography Results'''
 
With the use of a C18 reverse phase column we successfully separated Curcumin I, II, and III from the original solution. The base line resolution of the curcumin fractions wasadjusted by increasing the run time from the original C18 procedure from 15 min to 25 min.
 
[[Media:Curcumin Flash graph.pdf|Curcumin Flash Chromatography Image]]
 
 
 
::'''HPLC Results'''
 
having separated the three curcumin through flash chromatography, purity of the sample was tested using flash chromatography using the original solution as a basis of comparison.
 
[[File:T2 Curcumin HPLC chromatogram.jpg|500px|thumb|none|HPLC chromatogram]]
 
 
 
===6. 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.
 
  
===7. Conclusions===
+
:'''Rotovaporization''':
No final conclusions have been reached at this point, as we are still waiting to collect NMR results. The results of the NMR should tell us how successful our method was in isolating and purifying curcuminoids I,II, and III.
+
:: 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 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|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.]]
  
===8. Future Directions===
+
===Discussion===
We plan to continue this research project into the second semester of the 2016-17 school year (and beyond). First on our agenda will be to collect NMR data on the three separate curcuminoids that were rotovapped from the separate flash eluents. NMR data will be collected both at Monmouth College and Knox College. Results will indicate the success of our methods, and lead us to either additional method development or mass quantification using the latest method.
+
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.  
  
===9. Literature references===
+
===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
+
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===
 
===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.
 
Two copies of the report will be signed and dated and turned in to the Faculty Research Advisor and archived by the Research Coordinator.

Latest revision as of 15:34, 5 September 2019

You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project. This page was created by Stephanie Saey and Nadia Ayala. Nadia was a 2017 Biochemistry graduate and Stephanie was a 2018 Biochemistry/Biopsychology graduate.

Abstract

Turmeric, Curcuma longa, is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties. The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography. However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids (I/II/III) in amounts large enough for further investigation on its radicalisation properties.

The Three Curcuminoids

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:

Structures retrieved from: Curcuminoid Analogs with Potent Activity (Article)

Curcumin I, II, and III

Written Report

Descriptive information

Isolation and Purification of Curcuminoids from Tumeric Plant Curcuma Longa

Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon

Special thanks to Michael Prinsell and Broddie Davis

Research work documented in lab notebooks.

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.

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 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 1993;41:1640-3.

Reference
Methanol in Soxhlet Extraction:
1. Soxhlet apparatus was set up for extraction, as pictured in Image A.
2. Approximately 80 grams of organic turmeric from Starwest Botanicals was added to the thimble.
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.
Image A: soxhlet extraction
Removal of MeOH and Impurities:
1. MeOH/curcuminoid mixture in the stillpot (from extraction) was transferred to an 1000mL separatory funnel.
2. 275 mL of ethyl acetate (EtOAc) and 150 mL of water were added to the funnel, along with 150 mL brine solution.
3. The stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
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 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. The entire process resulted in approximately 250 mL of crude curcumin mixed with the solvents used for extraction.
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 Image C. Final Rf values were recorded.
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
Used rotovap (in Organic Chemistry lab) to concentrate the curcumin mixture before running Flash Chromatography.
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.
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.

Method is saved as "ssaeycurcuminsummer17" in the Flash Chromatography instrument.

Figure 1: Flash Chromatography separation of the crude curcumin extract. The first peak was presumed to be bidemethoxycurcumin (curcumin III), the most polar of the three curcuminoids, followed by demethoxycurcumin (curcumin II) and curcumin I (most nonpolar).
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.
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 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.
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.

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 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.

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

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

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.

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.