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Lignan Project

2021-05-10T19:25:06Z

Tcoffman: /* Abstract */

''The following document was completed by Taylor Coffman in Spring of 2021 in partial fulfillment of her requirements for a Bachelor's Degree of Science in Biochemistry, Monmouth College, Monmouth, Il 61462.''
==Abstract==
Lignin monomers can couple to form a large organic polymer or dimerize to form lignan. Lignan is a biologically active compound with various uses in the medical field, but lignan itself is extremely expensive for use in an undergraduate lab, with standards ranging in the upper three figures. An easily obtained product claiming to contain a certain lignan, hydroxymatairesinol, is available for purchase on the retailer Amazon.com for a low cost compared to commercially available standards. Purification, concentration, and eventual characterization of this material may lead to the availability of a lignan for use in undergraduate research labs. Use of HPLC and computational techniques (Gaussian and WINSIM) may lead to further understanding of the oxidative coupling of these monomers and could eventually lead to the commercial production of different lignans.

==Introduction==
Lignin is an abundant, complex organic polymer that acts as one of the principle components of plants. Lignin plays a role in the structure of plant cell walls, works to move water through the plant to critical areas and helps to prevent water evaporation.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> Lignins account for a large percentage of overall plant mass, at around 20-25%.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> We know that lignin does not act alone in it’s structural duties, commonly forming complexes with polymeric carbohydrates.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> Difficulty in isolating lignin from these lignin-carbohydrate complexes means that synthetic lignins are commonly used for research purposes. The basic monomers that make up the lignin polymer are p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These three monomers are phenylpropane units that differ in their substitutions at the 3 and 5 positions.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> In the case of lignin nomenclature, the side chain attachment to the aromatic ring is considered position C-1 and the phenol group considered to be position C-4. These monomeric alcohols undergo oxidative coupling with each other and with a growing polymer end unit.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> This oxidation process results in radicals delocalized at positions C-1, C-3, C-5, C-β, and O-4.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> The most frequently formed bonds are β-O-4 bonds.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref>

<gallery class="center">
File:P-coumaryl.jpg|''P-Coumaryl Alcohol''
File:coniferyl alc poster.png|''Coniferyl Alcohol''
File:sinapyl alc poster.png|''Sinapyl Alcohol''
</gallery>

The coupling of two lignin monomers, usually at their β carbons, results in lignan, a nonstructural component of plants that play a role in plant defense.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> These lignans show important biological activity that the lignin polymer does not.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> One interesting medical application of lignan is found in its interactions with breast cancer. It has been shown that both flaxseed and sesame seed lignans and their metabolites have the potential to act as competitive inhibitors of estrogen and prevent the binding of estrogen to estrogen receptors on breast cancer tumors.<ref>[[:Media:naturally lignan-rich foods.pdf|Rodríguez-García et al. (2019). Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion. ''Molecules, 24''(917), 1-25.]]</ref><ref>[[:Media:Estrogenic Activities of Sesame Lignans and Their Metabolites.pdf|Pianjing, P., Thiantanawat, A., Rangkadilok, N., Watcharasit, P., Mahidol, C., Satayavivad, J. (2001) ''Journal of Agricultural and Food Chemistry'', 59, 212–221]]</ref>. These effects are possible on the basis that many lignans share structural features with the human estrogen estradiol, including hydrophobic benzene rings and alcohol groups that are capable of interaction with the binding site of the estrogen receptor.<ref>[[:Media:Estrogenic Activities of Sesame Lignans and Their Metabolites.pdf|Pianjing, P., Thiantanawat, A., Rangkadilok, N., Watcharasit, P., Mahidol, C., Satayavivad, J. (2001) ''Journal of Agricultural and Food Chemistry'', 59, 212–221]]</ref><ref>[[:Media:Molecular basis of agonism and antagonism in the oestrogen receptor.pdf|Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engström, O., Ohman, L., Greene, G. L., Gustafsson, J. A., Carlquist, M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. ''Nature'' 389 (6652), 753–758.]]</ref> Lignan also displays antioxidant properties, showing the ability to scavenge hydroxyl radicals.<ref>[[:Media:naturally lignan-rich foods.pdf|Rodríguez-García et al. (2019). Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion. ''Molecules, 24''(917), 1-25.]]</ref><ref>[[:Media:Flaxseed Lignans.pdf|Touré A., Xu X. (2010). Flaxseed Lignans: Source, Biosynthesis, Metabolism, Antioxidant Activity, Bio-Active Components, Health Benefits. ''Comprehensive Reviews in Food Science and Food Safety'' 9:261–269.]]</ref> Lignan’s medical applications make it an interesting topic for further study, but an issue lies within the commercial availability of lignan. Lignan itself is not commercially available except as an analytical standard, and analytical standards can range upwards of $300 USD for 10 mg of product. For an undergraduate research lab, this presents serious problems in terms of budget and funding. In order to study the chemistry of lignans, we need a way to isolate and purify lignans in a cost effective manner.

==Experimental Methods==
===Materials===
Lignans for Life Hydroxymatairesinol (HMR) Lignan was obtained from Amazon.com (Seattle, WA, USA) while ethanol, 1,4-Dioxane, pH 5 Phosphate-Citrate Buffer, and KMnO4 were obtained from Sigma Aldrich (St. Louis, MO, USA).

===Lignan Purification===
Solutions of 1 HMR Lignan capsule (0.169 g), 5 HMR Lignan capsules (0.785 g), and 100.0 mL of EtOH and 10 HMR Lignan capsules (1.629 g) and 100.0 mL of EtOH were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Lignan Concentration===
Solutions of 2 HMR Lignan capsules + 20.0 mL of 1,4-Dioxane and 4 HMR Lignan capsules + 40.0 mL of 1,4-Dioxane were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

The solution of 4 HMR Lignan capsules in 40 mL of 1,4-Dioxane was used to fill ten 1.5 mL Eppendorf tubes with 1.0 mL of solution. These Eppendorf tubes were placed in a Savant Speedvac SC110 Concentrator (Holbrook, NY, USA) and spun overnight to remove the solvent. The pelleted solute in each Eppendorf tube was then resolubilized using 1.0 mL of EtOH.

The Lignan concentrate (10 μL) in 1.0 mL of H2O was ran on an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Lignan Degradation===
A solution of 10 HMR Lignan capsules in 100.0 mL of 1,4-Dioxane solution was prepared. This HMR Lignan/dioxane solution (25.0 mL) was added to 25.0 mL of pH 5 Phosphate-Citrate buffer.

A solution of 0.0395 g of KMnO4 and 1000 μL H2O was prepared.

The following solutions were analyzed on an Agilent 1100 HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA:
*5.0 mL of the HMR Lignan/dioxane/buffer solution
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 5 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 10 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 15 mMol KMnO4

===Electron Density Analysis of Lignin Monomer-Like Molecules===
Geometry Optimization and Molecular Orbital calculations were performed on Phenoxyl and 5-Vinylphenoxyl in WebMO using the B3YLP theory, accurate: 6-311+G(2d,p) basis set, and a density=current keyword. Hyperfine coupling constants were enteres into WINSIM in order to predict EPR Spectra.

==Results and Discussion==
===Lignan Purification===
[[File:lignanpurification.png|450px|thumb|center|Figure 1: Offset HPLC analysis of 1, 5, and 10 HMR Lignan capsules in 100.0 mL of ethanol. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

Lignan purification trials indicated that HMR Lignans already came in a relatively pure form. In Figure 1., each subsequent peak indicates that as the amount of solute increased per trial, the amount of "lignan" in the solution also increased, which indicates that the capsules are homogenous in terms of amount of material. There is also a low amount of noise, indicating that whatever material is in these capsules, the material is for mostly pure with little pollution. Further tests should be done to characterize this material and confirm that it is truly hydroxymatairesinol and to confirm that the manufacturers claims are accurate.

===Lignan Concentration===
{|
|-
||[[File:lignanconcentration1.png|450px|thumb|Figure 2: Offset HPLC analysis of 2 HMR Lignan Capsules in 20.0 mL of 1,4-Dioxane and 4 HMR Lignan Capsules in 40.0 mL of 1,4-Dioxane. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]] || [[File:1uL in 1mL h20.png|450px|thumb|Figure 3: HPLC analysis of 10.0 μL of Lignan concentrate in 1.0 mL of H2O. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]
|}

In order to get a workable amount of lignan from these capsules, concentration was performed in order to produce enough product so that further studies can be performed using this material. This could include characterization of the product and assays to determine if the material has antibiotic properties. Figure 2. indicates that there is a relatively consistent amount of material in these capsules and that there is some level of quality control of the overall product. This is important while performing concentration of the product so that a reliable amount of concentrate can be obtained. Figure 3. indicates that that material was successfully concentrated. Done at a larger scale, this portion of the overall project will be helpful towards gaining a tangible amount of material for further testing.

===Lignan Degradation===
[[File:lignandegradation.png||450px|thumb|center|Figure 4: Offset HPLC Analysis of 5.0 mL of HMR Lignan/dioxane/buffer solution and various amounts of KMnO4. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

In order to better understand the coupling of lignin monomers, degradation of lignan can be performed. Understanding the modes of the degradation of lignan can help understand the pattern in which lignin monomers couple. In Figure 4., we see that with increased amount of KMnO4, the amount of starting material decreases and the amount of products increases, indicating that successful degradation of this material was completed. Further analysis of these degradation products, for example through characterization of theese products, may allow better insight into the coupling of lignin monomers.

===Electron Density Analysis of Lignin Monomer-Like Molecules===
{|
|-
||[[File:TCMolecule1.png|450px|thumb|Figure 5. Phenoxyl]] || [[File:TCMolecule2.png|450px|thumb|Figure 6. 4-Vinylphenoxyl]]
|-
||[[File:molecule1edensity.png|450px|thumb|Figure 7. Radical Frontier Density Diagram of Phenoxyl]] || [[File:molecule2edensity.png|450px|thumb|Figure 8. Radical Frontier Density Diagram of 4-Vinylphenoxyl]]
|-
||[[File:molecule1esr.png|450px|thumb|Figure 9. Simulated ESR Spectrum of Phenoxyl]] || [[File:Molecule2esr.png|450px|thumb|Figure 10. Simulated ESR Spectrum of 4-Vinylphenoxyl]]
|}
Table 1. Hyperfine coupling constants of Phenoxy and 4-Vinylphenoxy
{| class="wikitable"
|-
! Phenoxyl !! 4-Vinylphenoxyl
|-
|2.65 || 2.33
|-
| 2.65 || 2.50
|-
| 6.86 || 2.70
|-
| 6.68 || 5.35
|-
| 9.12 || 6.24
|-
| || 6.72
|-
| || 7.17
|}

Figures 5-9. illustrate the structure, radical frontier density, and predicted ESR spectrum of two lignin monomer-like structures, Phenoxyl and 4-Vinylphenoxyl. Table 1. lists the hyperfine coupling constants used to construct the predicted ESR spectrum. The radical density of these structures (blue/green areas) allows us to predict that the radical would spend the majority of it's time in the ortho and para positions. While Phenoxyl and 4-Vinylphenoxyl are not lignin monomers, there structures are similar to those of the three lignin monomers (p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol) and can give us insight into how these monomers radicalize and couple to form both lignan and lignin. If these reactions are better understood, lignan itself could be synthetically produced for use in various medical applications.

==Conclusion==
In terms of studying lignin, lignan, and lignin monomers in an undergraduate laboratory, cost and availability of materials must be strongly considered. A cheap source of a potential lignan, hydroxymatairesinol, can be obtained from Amazon.com in the form of a human supplement. Purification and concentration of the material may lead to a cheap source of HMR lignan that can be utilized in undergraduate laboratories for potential projects relating to the coupling of lignin monomers or the degradation of lignin. As lignan itself is known to have biological activity, accessible amount of HMR lignan can be utilized in a lab to run different biological tests for antibacterial, antimicrobial, and anticancer activity. There is a lot of work to be done in the understanding of lignin monomer coupling, which is highly randomized and not well understood. Because as of now these studies are not cost effective for an undergraduate lab, being able to access cheap lignan will improve the viability of this research. This research project has indicated that obtainment of cheap hydroxymatairesinol from this readily available source is not only possible, but that this method may be used towards purification of other lignans from products marketed as supplements.

==References==

Taylor Coffman

2021-05-09T16:06:56Z

Tcoffman: /* Career Plans */

You have reached the personal page of Taylor Coffman.

==Personal Information==
Senior Biochemistry Major

Hometown: Carpentersville, IL

==Undergraduate Research Activities==

====Spring 2020====

*Worked as mentee of [http://esr.monmsci.net/wiki/index.php/Zelinda_Taylor ''Zelinda Taylor'']

====Fall 2020 and Spring 2021====

[[Lignan Project|Compiled Lignan Project]]
*[[Lignan Purification|Lignan Purification]]
*[[Lignan Concentration|Lignan Concentration]]
*[[Lignan Degradation|Lignan Degradation]]
*[[Electron Density of Phenoxyl and 4-Vinylphenoxyl|Electron Density of Phenoxyl and 4-Vinylphenoxyl]]

==Career Plans==
*Apply to medical school for the fall of 2022

Taylor Coffman

2021-05-09T16:06:32Z

Tcoffman:

2021-05-08T23:44:46Z

Tcoffman: /* Results */

==Abstract==

==Introduction==
Lignin is an abundant, complex organic polymer that acts as one of the principle components of plants. Lignin plays a role in the structure of plant cell walls, works to move water through the plant to critical areas and helps to prevent water evaporation.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> Lignins account for a large percentage of overall plant mass, at around 20-25%.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> We know that lignin does not act alone in it’s structural duties, commonly forming complexes with polymeric carbohydrates.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> Difficulty in isolating lignin from these lignin-carbohydrate complexes means that synthetic lignins are commonly used for research purposes. The basic monomers that make up the lignin polymer are p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These three monomers are phenylpropane units that differ in their substitutions at the 3 and 5 positions.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> In the case of lignin nomenclature, the side chain attachment to the aromatic ring is considered position C-1 and the phenol group considered to be position C-4. These monomeric alcohols undergo oxidative coupling with each other and with a growing polymer end unit.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> This oxidation process results in radicals delocalized at positions C-1, C-3, C-5, C-β, and O-4.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> The most frequently formed bonds are β-O-4 bonds.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref>

<gallery class="center">
File:P-coumaryl.jpg|''P-Coumaryl Alcohol''
File:coniferyl alc poster.png|''Coniferyl Alcohol''
File:sinapyl alc poster.png|''Sinapyl Alcohol''
</gallery>

The coupling of two lignin monomers, usually at their β carbons, results in lignan, a nonstructural component of plants that play a role in plant defense.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> These lignans show important biological activity that the lignin polymer does not.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> One interesting medical application of lignan is found in its interactions with breast cancer. It has been shown that both flaxseed and sesame seed lignans and their metabolites have the potential to act as competitive inhibitors of estrogen and prevent the binding of estrogen to estrogen receptors on breast cancer tumors.<ref>[[:Media:naturally lignan-rich foods.pdf|Rodríguez-García et al. (2019). Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion. ''Molecules, 24''(917), 1-25.]]</ref><ref>[[:Media:Estrogenic Activities of Sesame Lignans and Their Metabolites.pdf|Pianjing, P., Thiantanawat, A., Rangkadilok, N., Watcharasit, P., Mahidol, C., Satayavivad, J. (2001) ''Journal of Agricultural and Food Chemistry'', 59, 212–221]]</ref>. These effects are possible on the basis that many lignans share structural features with the human estrogen estradiol, including hydrophobic benzene rings and alcohol groups that are capable of interaction with the binding site of the estrogen receptor.<ref>[[:Media:Estrogenic Activities of Sesame Lignans and Their Metabolites.pdf|Pianjing, P., Thiantanawat, A., Rangkadilok, N., Watcharasit, P., Mahidol, C., Satayavivad, J. (2001) ''Journal of Agricultural and Food Chemistry'', 59, 212–221]]</ref><ref>[[:Media:Molecular basis of agonism and antagonism in the oestrogen receptor.pdf|Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engström, O., Ohman, L., Greene, G. L., Gustafsson, J. A., Carlquist, M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. ''Nature'' 389 (6652), 753–758.]]</ref> Lignan also displays antioxidant properties, showing the ability to scavenge hydroxyl radicals.<ref>[[:Media:naturally lignan-rich foods.pdf|Rodríguez-García et al. (2019). Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion. ''Molecules, 24''(917), 1-25.]]</ref><ref>[[:Media:Flaxseed Lignans.pdf|Touré A., Xu X. (2010). Flaxseed Lignans: Source, Biosynthesis, Metabolism, Antioxidant Activity, Bio-Active Components, Health Benefits. ''Comprehensive Reviews in Food Science and Food Safety'' 9:261–269.]]</ref> Lignan’s medical applications make it an interesting topic for further study, but an issue lies within the commercial availability of lignan. Lignan itself is not commercially available except as an analytical standard, and analytical standards can range upwards of $300 USD for 10 mg of product. For an undergraduate research lab, this presents serious problems in terms of budget and funding. In order to study the chemistry of lignans, we need a way to isolate and purify lignans in a cost effective manner.

==Experimental Methods==
===Materials===
Lignans for Life Hydroxymatairesinol (HMR) Lignan was obtained from Amazon.com (Seattle, WA, USA) while ethanol, 1,4-Dioxane, pH 5 Phosphate-Citrate Buffer, and KMnO4 were obtained from Sigma Aldrich (St. Louis, MO, USA).

===Lignan Purification===
Solutions of 1 HMR Lignan capsule (0.169 g), 5 HMR Lignan capsules (0.785 g), and 100.0 mL of EtOH and 10 HMR Lignan capsules (1.629 g) and 100.0 mL of EtOH were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Lignan Concentration===
Solutions of 2 HMR Lignan capsules + 20.0 mL of 1,4-Dioxane and 4 HMR Lignan capsules + 40.0 mL of 1,4-Dioxane were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

The solution of 4 HMR Lignan capsules in 40 mL of 1,4-Dioxane was used to fill ten 1.5 mL Eppendorf tubes with 1.0 mL of solution. These Eppendorf tubes were placed in a Savant Speedvac SC110 Concentrator (Holbrook, NY, USA) and spun overnight to remove the solvent. The pelleted solute in each Eppendorf tube was then resolubilized using 1.0 mL of EtOH.

The Lignan concentrate (10 μL) in 1.0 mL of H2O was ran on an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Lignan Degradation===
A solution of 10 HMR Lignan capsules in 100.0 mL of 1,4-Dioxane solution was prepared. This HMR Lignan/dioxane solution (25.0 mL) was added to 25.0 mL of pH 5 Phosphate-Citrate buffer.

A solution of 0.0395 g of KMnO4 and 1000 μL H2O was prepared.

The following solutions were analyzed on an Agilent 1100 HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA:
*5.0 mL of the HMR Lignan/dioxane/buffer solution
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 5 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 10 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 15 mMol KMnO4

===Electron Density Analysis of Lignin Monomer-Like Molecules===
Geometry Optimization and Molecular Orbital calculations were performed on Phenoxyl and 5-Vinylphenoxyl in WebMO using the B3YLP theory, accurate: 6-311+G(2d,p) basis set, and a density=current keyword. Hyperfine coupling constants were enteres into WinSim in order to predict EPR Spectra

==Results==
===Lignan Purification===
[[File:lignanpurification.png|450px|thumb|center|Figure 1: Offset HPLC analysis of 1, 5, and 10 HMR Lignan capsules in 100.0 mL of ethanol. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

===Lignan Concentration===
{|
|-
||[[File:lignanconcentration1.png|450px|thumb|Figure 2: Offset HPLC analysis of 2 HMR Lignan Capsules in 20.0 mL of 1,4-Dioxane and 4 HMR Lignan Capsules in 40.0 mL of 1,4-Dioxane. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]] || [[File:1uL in 1mL h20.png|450px|thumb|Figure 3: HPLC analysis of 10.0 μL of Lignan concentrate in 1.0 mL of H2O. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]
|-
|}

===Lignan Degradation===
[[File:lignandegradation.png||450px|thumb|center|Figure 4: Offset HPLC Analysis of 5.0 mL of HMR Lignan/dioxane/buffer solution and various amounts of KMnO4. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

===Electron Density Analysis of Lignin Monomer-Like Molecules===
{|
|-
||[[File:TCMolecule1.png|450px|thumb|Figure 5. Phenoxyl]] || [[File:TCMolecule2.png|450px|thumb|Figure 6. 4-Vinylphenoxyl]]
|-
||[[File:molecule1edensity.png|450px|thumb|Figure 7. Radical Frontier Density Diagram of Phenoxyl]] || [[File:molecule2edensity.png|450px|thumb|Figure 8. Radical Frontier Density Diagram of 4-Vinylphenoxyl]]
|-
||[[File:molecule1esr.png|450px|thumb|Figure 9. Simulated ESR Spectrum of Phenoxyl]] || [[File:Molecule2esr.png|450px|thumb|Figure 10. Simulated ESR Spectrum of 4-Vinylphenoxyl]]
|}
====Hyperfine Coupling Constants====
{| class="wikitable"
|-
! Phenoxy !! 4-Vinylphenoxy
|-
|2.65 || 2.33
|-
| 2.65 || 2.5
|-
| 6.86 || 2.7
|-
| 6.68 || 5.35
|-
| 9.12 || 6.24
|-
| || 6.72
|-
| || 7.17
|}

==Discussion==

==Conclusion==

==References==

Lignan Project

2021-05-08T23:40:10Z

Tcoffman: /* Results */

==Abstract==

==Introduction==
Lignin is an abundant, complex organic polymer that acts as one of the principle components of plants. Lignin plays a role in the structure of plant cell walls, works to move water through the plant to critical areas and helps to prevent water evaporation.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> Lignins account for a large percentage of overall plant mass, at around 20-25%.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> We know that lignin does not act alone in it’s structural duties, commonly forming complexes with polymeric carbohydrates.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> Difficulty in isolating lignin from these lignin-carbohydrate complexes means that synthetic lignins are commonly used for research purposes. The basic monomers that make up the lignin polymer are p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These three monomers are phenylpropane units that differ in their substitutions at the 3 and 5 positions.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> In the case of lignin nomenclature, the side chain attachment to the aromatic ring is considered position C-1 and the phenol group considered to be position C-4. These monomeric alcohols undergo oxidative coupling with each other and with a growing polymer end unit.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> This oxidation process results in radicals delocalized at positions C-1, C-3, C-5, C-β, and O-4.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref> The most frequently formed bonds are β-O-4 bonds.<ref>[[:Media:Dimmel Overview.pdf|Dimmel, D. (2010). Overview. ''Lignin and Lignans Advances in Chemistry.'' Taylor & Francis Group.]]</ref>

<gallery class="center">
File:P-coumaryl.jpg|''P-Coumaryl Alcohol''
File:coniferyl alc poster.png|''Coniferyl Alcohol''
File:sinapyl alc poster.png|''Sinapyl Alcohol''
</gallery>

The coupling of two lignin monomers, usually at their β carbons, results in lignan, a nonstructural component of plants that play a role in plant defense.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> These lignans show important biological activity that the lignin polymer does not.<ref>[[:Media:Lignan and Lignan Biosynthesis.pdf|Lewis, N. G., Davin, L. B., & Sarkanen, S. (1998) Lignan and Lignan Biosynthesis: Distinctions and Reconciliations. ''American Chemical Society Symposium Series'', 1-27.]]</ref> One interesting medical application of lignan is found in its interactions with breast cancer. It has been shown that both flaxseed and sesame seed lignans and their metabolites have the potential to act as competitive inhibitors of estrogen and prevent the binding of estrogen to estrogen receptors on breast cancer tumors.<ref>[[:Media:naturally lignan-rich foods.pdf|Rodríguez-García et al. (2019). Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion. ''Molecules, 24''(917), 1-25.]]</ref><ref>[[:Media:Estrogenic Activities of Sesame Lignans and Their Metabolites.pdf|Pianjing, P., Thiantanawat, A., Rangkadilok, N., Watcharasit, P., Mahidol, C., Satayavivad, J. (2001) ''Journal of Agricultural and Food Chemistry'', 59, 212–221]]</ref>. These effects are possible on the basis that many lignans share structural features with the human estrogen estradiol, including hydrophobic benzene rings and alcohol groups that are capable of interaction with the binding site of the estrogen receptor.<ref>[[:Media:Estrogenic Activities of Sesame Lignans and Their Metabolites.pdf|Pianjing, P., Thiantanawat, A., Rangkadilok, N., Watcharasit, P., Mahidol, C., Satayavivad, J. (2001) ''Journal of Agricultural and Food Chemistry'', 59, 212–221]]</ref><ref>[[:Media:Molecular basis of agonism and antagonism in the oestrogen receptor.pdf|Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engström, O., Ohman, L., Greene, G. L., Gustafsson, J. A., Carlquist, M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. ''Nature'' 389 (6652), 753–758.]]</ref> Lignan also displays antioxidant properties, showing the ability to scavenge hydroxyl radicals.<ref>[[:Media:naturally lignan-rich foods.pdf|Rodríguez-García et al. (2019). Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion. ''Molecules, 24''(917), 1-25.]]</ref><ref>[[:Media:Flaxseed Lignans.pdf|Touré A., Xu X. (2010). Flaxseed Lignans: Source, Biosynthesis, Metabolism, Antioxidant Activity, Bio-Active Components, Health Benefits. ''Comprehensive Reviews in Food Science and Food Safety'' 9:261–269.]]</ref> Lignan’s medical applications make it an interesting topic for further study, but an issue lies within the commercial availability of lignan. Lignan itself is not commercially available except as an analytical standard, and analytical standards can range upwards of $300 USD for 10 mg of product. For an undergraduate research lab, this presents serious problems in terms of budget and funding. In order to study the chemistry of lignans, we need a way to isolate and purify lignans in a cost effective manner.

==Experimental Methods==
===Materials===
Lignans for Life Hydroxymatairesinol (HMR) Lignan was obtained from Amazon.com (Seattle, WA, USA) while ethanol, 1,4-Dioxane, pH 5 Phosphate-Citrate Buffer, and KMnO4 were obtained from Sigma Aldrich (St. Louis, MO, USA).

===Lignan Purification===
Solutions of 1 HMR Lignan capsule (0.169 g), 5 HMR Lignan capsules (0.785 g), and 100.0 mL of EtOH and 10 HMR Lignan capsules (1.629 g) and 100.0 mL of EtOH were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Lignan Concentration===
Solutions of 2 HMR Lignan capsules + 20.0 mL of 1,4-Dioxane and 4 HMR Lignan capsules + 40.0 mL of 1,4-Dioxane were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

The solution of 4 HMR Lignan capsules in 40 mL of 1,4-Dioxane was used to fill ten 1.5 mL Eppendorf tubes with 1.0 mL of solution. These Eppendorf tubes were placed in a Savant Speedvac SC110 Concentrator (Holbrook, NY, USA) and spun overnight to remove the solvent. The pelleted solute in each Eppendorf tube was then resolubilized using 1.0 mL of EtOH.

The Lignan concentrate (10 μL) in 1.0 mL of H2O was ran on an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Lignan Degradation===
A solution of 10 HMR Lignan capsules in 100.0 mL of 1,4-Dioxane solution was prepared. This HMR Lignan/dioxane solution (25.0 mL) was added to 25.0 mL of pH 5 Phosphate-Citrate buffer.

A solution of 0.0395 g of KMnO4 and 1000 μL H2O was prepared.

The following solutions were analyzed on an Agilent 1100 HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA:
*5.0 mL of the HMR Lignan/dioxane/buffer solution
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 5 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 10 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 15 mMol KMnO4

===Electron Density Analysis of Lignin Monomer-Like Molecules===
Geometry Optimization and Molecular Orbital calculations were performed on Phenoxyl and 5-Vinylphenoxyl in WebMO using the B3YLP theory, accurate: 6-311+G(2d,p) basis set, and a density=current keyword. Hyperfine coupling constants were enteres into WinSim in order to predict EPR Spectra

==Results==
===Lignan Purification===
[[File:lignanpurification.png|450px|thumb|center|Figure 1: Offset HPLC analysis of 1, 5, and 10 HMR Lignan capsules in 100.0 mL of ethanol. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

===Lignan Concentration===
{|
|-
||[[File:lignanconcentration1.png|450px|thumb|Figure 1: Offset HPLC analysis of 2 HMR Lignan Capsules in 20.0 mL of 1,4-Dioxane and 4 HMR Lignan Capsules in 40.0 mL of 1,4-Dioxane. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]] || [[File:1uL in 1mL h20.png|450px|thumb|Figure 1: HPLC analysis of 10.0 μL of Lignan concentrate in 1.0 mL of H2O. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]
|-
|}

===Lignan Degradation===
[[File:lignandegradation.png||450px|thumb|center|Figure 1: Offset HPLC Analysis of 5.0 mL of HMR Lignan/dioxane/buffer solution and various amounts of KMnO4. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

===Electron Density Analysis of Lignin Monomer-Like Molecules===
{|
|-
||[[File:TCMolecule1.png|450px|thumb|Phenoxyl]] || [[File:TCMolecule2.png|450px|thumb|4-Vinylphenoxyl]]
|-
||[[File:molecule1edensity.png|450px|thumb|Radical Frontier Density Diagram of Phenoxyl]] || [[File:molecule2edensity.png|450px|thumb|Radical Frontier Density Diagram of 4-Vinylphenoxyl]]
|-
||[[File:molecule1esr.png|450px|thumb|Simulated ESR Spectrum of Phenoxyl]] || [[File:Molecule2esr.png|450px|thumb|Simulated ESR Spectrum of 4-Vinylphenoxyl]]
|}
====Hyperfine Coupling Constants====
{| class="wikitable"
|-
! Phenoxy !! 4-Vinylphenoxy
|-
|2.65 || 2.33
|-
| 2.65 || 2.5
|-
| 6.86 || 2.7
|-
| 6.68 || 5.35
|-
| 9.12 || 6.24
|-
| || 6.72
|-
| || 7.17
|}

==Discussion==

==Conclusion==

==References==

Lignan Project

2021-05-08T23:39:07Z

Tcoffman: /* Experimental Methods */

Taylor Coffman

2021-05-08T23:35:43Z

Tcoffman: /* Undergraduate Research Activities */

You have reached the personal page of Taylor Coffman.

==Personal Information==
Senior Biochemistry Major

Hometown: Carpentersville, IL

==Undergraduate Research Activities==

Spring 2020

*Worked as mentee of [http://esr.monmsci.net/wiki/index.php/Zelinda_Taylor ''Zelinda Taylor'']
*[http://esr.monmsci.net/wiki/index.php/Oxidative_Properties_of_Lignan ''Biological Dimers: Lignan'']

Fall 2020 and Spring 2021
*[http://esr.monmsci.net/wiki/index.php/Lignan_Project ''Lignan Project'']
*[http://esr.monmsci.net/wiki/index.php/Lignan_Purification ''Lignan Purification'']
*[http://esr.monmsci.net/wiki/index.php/Lignan_Concentration ''Lignan Concentration'']
*[http://esr.monmsci.net/wiki/index.php/Lignan_Degradation ''Lignan Degradation'']

Taylor Coffman

2021-05-05T20:50:58Z

Tcoffman: /* Undergraduate Research Activities */

You have reached the personal page of Taylor Coffman.

==Personal Information==
Senior Biochemistry Major

Hometown: Carpentersville, IL

==Undergraduate Research Activities==

Spring 2020

*Worked as mentee of [http://esr.monmsci.net/wiki/index.php/Zelinda_Taylor ''Zelinda Taylor'']
*[http://esr.monmsci.net/wiki/index.php/Oxidative_Properties_of_Lignan ''Biological Dimers: Lignan'']

Fall 2020 and Spring 2021
{| class="wikitable"
|-
|*[http://esr.monmsci.net/wiki/index.php/Lignan_Project ''Lignan Project'']
|-
| ||*[http://esr.monmsci.net/wiki/index.php/Lignan_Purification ''Lignan Purification'']
|-
| ||*[http://esr.monmsci.net/wiki/index.php/Lignan_Concentration ''Lignan Concentration'']
|-
*[http://esr.monmsci.net/wiki/index.php/Lignan_Degradation ''Lignan Degradation'']
|}

Taylor Coffman

2021-05-05T20:49:38Z

Tcoffman: /* Undergraduate Research Activities */

You have reached the personal page of Taylor Coffman.

==Personal Information==
Senior Biochemistry Major

Hometown: Carpentersville, IL

==Undergraduate Research Activities==

Spring 2020

*Worked as mentee of [http://esr.monmsci.net/wiki/index.php/Zelinda_Taylor ''Zelinda Taylor'']
*[http://esr.monmsci.net/wiki/index.php/Oxidative_Properties_of_Lignan ''Biological Dimers: Lignan'']

Fall 2020 and Spring 2021
{|
|-
| ||*[http://esr.monmsci.net/wiki/index.php/Lignan_Project ''Lignan Project'']
|-
| ||*[http://esr.monmsci.net/wiki/index.php/Lignan_Purification ''Lignan Purification'']
|-
| ||*[http://esr.monmsci.net/wiki/index.php/Lignan_Concentration ''Lignan Concentration'']
|-
*[http://esr.monmsci.net/wiki/index.php/Lignan_Degradation ''Lignan Degradation'']
|}

Lignan Degradation

2021-05-05T14:51:09Z

Tcoffman: /* Materials and Methods */

===Materials and Methods===
Lignans for Life Hydroxymatairesinol (HMR) Lignan was obtained from Amazon.com (Seattle, WA, USA), and pH 5 Phosphate-Citrate Buffer, 1,4-Dioxane, and KMnO4 and were obtained from Sigma Aldrich (St. Louis, MO, USA).

A solution of 10 HMR Lignan capsules in 100.0 mL of 1,4-Dioxane solution was prepared. This HMR Lignan/dioxane solution (25.0 mL) was added to 25.0 mL of pH 5 Phosphate-Citrate buffer.

A solution of 0.0395 g of KMnO4 and 1000 μL H2O was prepared.

The following solutions were analyzed on an Agilent 1100 HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA:
*5.0 mL of the HMR Lignan/dioxane/buffer solution
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 5 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 10 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 15 mMol KMnO4

===Results and Discussion===
[[File:lignandegradation.png||450px|thumb|center|Figure 1: Offset HPLC Analysis of 5.0 mL of HMR Lignan/dioxane/buffer solution and various amounts of KMnO4. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

Lignan Degradation

2021-05-05T14:48:51Z

Tcoffman: /* Materials and Methods */

===Materials and Methods===
Lignans for Life Hydroxymatairesinol (HMR) Lignan was obtained from Amazon.com (Seattle, WA, USA), and pH 5 Phosphate-Citrate Buffer, 1,4-Dioxane, and KMnO4 and were obtained from Sigma Aldrich (St. Louis, MO, USA).

A solution of 10 HMR Lignan capsules in 100.0 mL of 1,4-Dioxane solution was prepared. This HMR Lignan/dioxane solution (25.0 mL) was added to 25.0 mL of pH 5 Phosphate-Citrate buffer.

A solution of 0.0395 g of KMnO4 and 1000 μL H2O was prepared.

The following solutions were analyzed on an Agilent 1100 HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA:
*5.0 mL of the HMR Lignan/dioxane/buffer solution
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 5 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 10 mMol KMnO4
*5.0 mL of the HMR Lignan/dioxane/buffer solution + 15 mMol KMnO4

==Materials and Methods==
[[File:lignandegradation.png||450px|thumb|center|Figure 1: Offset HPLC Analysis of 5.0 mL of HMR Lignan/dioxane/buffer solution and various amounts of KMnO4. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

Lignan Purification

2021-05-05T14:48:21Z

Tcoffman: /* Results and Discussion */

==Lignan Purification==
===Materials and Methods===
Lignans for Life Hydroxymatairesinol (HMR) Lignan was obtained from Amazon.com (Seattle, WA, USA) and ethanol was obtained from Sigma Aldrich (St. Louis, MO, USA).

Solutions of 1 HMR Lignan capsule (0.169 g), 5 HMR Lignan capsules (0.785 g), and 100.0 mL of EtOH and 10 HMR Lignan capsules (1.629 g) and 100.0 mL of EtOH were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Results and Discussion===
[[File:lignanpurification.png|450px|thumb|center|Figure 1: Offset HPLC analysis of 1, 5, and 10 HMR Lignan capsules in 100.0 mL of ethanol. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

The purification of lignan using an easily accessible source such as Lignans for Life’s HMR Lignans seems to be a promising method of gaining lignan for experimental use. Purification of these HMR lignan capsules produced a chromatogram that has a single peak, indicating that our sample is a relatively pure compound with little pollution (Figure 1.). As the absorption of each peak increases with higher mass of solute, we see that the amount of uncharacterized compound in the HMR Lignan capsules is relatively homogeneous across the samples. Because we cannot be sure that these HMR Lignan capsules contain hydroxymatairesinol, further characterization steps will need to be taken to ensure that the claims of the manufacturers are accurate.

===Future Directions===
Because this project has not reached any conclusion as to whether Lignans for Life HMR Lignan truly contains hydroxymatairesinol, further characterization of this powder must be performed. This can be done through nuclear magnetic resonance or through mass spectroscopy. Once a characterization of this unknown is completed, different tests can be performed in order to better understand the biological activity of this specific lignan, including a Kirby-Bauer test. If this lignan is easily isolated, this could also provide material for further lignin/lignan degradation and synthesis work.

Lignan Purification

2021-05-05T14:48:00Z

Tcoffman: /* Results and Discussion */

==Lignan Purification==
===Materials and Methods===
Lignans for Life Hydroxymatairesinol (HMR) Lignan was obtained from Amazon.com (Seattle, WA, USA) and ethanol was obtained from Sigma Aldrich (St. Louis, MO, USA).

Solutions of 1 HMR Lignan capsule (0.169 g), 5 HMR Lignan capsules (0.785 g), and 100.0 mL of EtOH and 10 HMR Lignan capsules (1.629 g) and 100.0 mL of EtOH were prepared. These solutions were filtered and analyzed using an Agilent 1100 Series HPLC with a C18 column (4.16 x 100 mm length and 3.5 μm particle size) at absorbance wavelength of 270 nm and temperature of 28°C using an acetonitrile (ACN) and 0.1% TFA gradient for 30 minutes. The first 15 minutes were run at 100% 0.1% TFA, 15-25 minutes at 100% ACN, and 25-30 minutes at 100% 0.1% TFA.

===Results and Discussion===
<gallery>
</gallery>[[File:lignanpurification.png|450px|thumb|center|Figure 1: Offset HPLC analysis of 1, 5, and 10 HMR Lignan capsules in 100.0 mL of ethanol. The sample was run through an Agilent 1100 HPLC with a C18 column (length: 4.6 x 100 mm and particle size: 3.5 μm) at 28.0°C with a gradient elution at 0-15 min: 100% 0.1% TFA, 0% acetonitrile, 15-25 min: 0% 0.1% TFA, 100% acetonitrile, 25-30 min: 100% 0.1% TFA, 0% acetonitrile. Trials were run once.]]

The purification of lignan using an easily accessible source such as Lignans for Life’s HMR Lignans seems to be a promising method of gaining lignan for experimental use. Purification of these HMR lignan capsules produced a chromatogram that has a single peak, indicating that our sample is a relatively pure compound with little pollution (Figure 1.). As the absorption of each peak increases with higher mass of solute, we see that the amount of uncharacterized compound in the HMR Lignan capsules is relatively homogeneous across the samples. Because we cannot be sure that these HMR Lignan capsules contain hydroxymatairesinol, further characterization steps will need to be taken to ensure that the claims of the manufacturers are accurate.

===Future Directions===
Because this project has not reached any conclusion as to whether Lignans for Life HMR Lignan truly contains hydroxymatairesinol, further characterization of this powder must be performed. This can be done through nuclear magnetic resonance or through mass spectroscopy. Once a characterization of this unknown is completed, different tests can be performed in order to better understand the biological activity of this specific lignan, including a Kirby-Bauer test. If this lignan is easily isolated, this could also provide material for further lignin/lignan degradation and synthesis work.

Lignan Project

2021-05-05T14:47:18Z

Tcoffman: /* Lignan Purification */