Curcumin Research

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.