Ethan B. Baxter Telephone: (816) 805-7889 Email: ethan.baxter@mail.missouri.edu Nathan D. Smith Telephone: (816) 824-4093 Email: ndsf3f@mail.missouri.edu

Department of Chemistry University of Missouri – Columbia

Chemistry Building 601 South College Avenue

Columbia, MO 65211 USA

________________________________________________________________________

April 29, 2014 Professor Rainer Glaser, Associate Editor 321 Chemistry Building 601 S. College Avenue Columbia, MO 65211 RE: REVISED

Alpha-linolenic acid in dietary chia seeds (Salvia hispanica) reduce effects of high sucrose diets

By Ethan Baxter and Nathan Smith Dear Dr. Glaser,

Thank you very much for considering our manuscript for publication. We appreciate the constructive criticism from the three reviewers. In the following, we are addressing the reviewer comments and describe all revisions made.

Major Revisions [M.1] The graphical abstract was edited to include a more distinct picture of chia seeds and to include a label for the structure of alpha-linolenic acid. [M.2] A scheme of alpha-linolenic acid’s structure was added to the Introduction section.

[M.3] The previous Scheme 1, illustrating the apparatus used in extraction of alpha-linolenic acid, was moved to Supporting Information as Scheme S1. The specifics of this apparatus are explained more in this section than in the body of the main text. [M.4] Table 1, which illustrates the 3 groups that were tested, as well as the metabolites and enzymes that were tested for, was moved to Results and Discussion.

Response to Reviewer 1 [1.1] We reordered the reference numbers so that they appear in chronological order.

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[1.2] Some examples of genetic diseases were inserted. [1.3] Typo on p. 10 corrected, the sentence now reads “Malic enzyme was decreased, but not to the level of the CD.” [1.4] We accepted to suggested correction by the reviewer and also improved more grammar in this sentence. It now reads, “The chia seed oil is collected through the extraction described.”

[1.5] Replaced “A” by “The” as suggested. The opening sentence of the abstract has also been altered slightly.

[1.6] The reviewer suggests to “Define dyslipidemia just so the reader better understands what it is if they encounter it later on in the paper.” We agree with the reviewer that this term should be defined, but we feel it is better served in the introduction rather than the abstract.

[1.7] A scheme of ALA was included as part of [M.2]. [1.8] We accepted this suggestion and clarified this sentence; it now reads “Unsaturated fatty acids are one specific type of nutraceutical.” [1.9] We understand the point the reviewer makes here, and because rats are the test subjects instead of mice, we exchanged the word “organisms” for “rats.” [1.10] The reviewer expressed concern with the wording of the sentence beginning with, “Here we report…” and said it should instead start with, “Here we test if…” possibly to signify that we were unsure about the status of the results this early in the paper. However, since we have already received results from our procedure, we are not still producing tests to see if chia seeds produce an effect on sucrose-rich diets – we have already determined that they do within the confines of this procedure. Further, our sentence reads, “Here we report whether feeding chia seeds… produces any effect.” Had we not found any effect of chia seeds on sucrose-rich diets in rats, we would have either stated this observation in our Discussion section or hesitated to write this manuscript due to the possibility of incomplete or inconclusive data. [1.11] This review statement appears to be the same as [1.9] because there are no other incidents of the word “organisms” in the text. In either case, this matter has been addressed.

[1.12] Some of the timid phrasing has been addressed and altered to sound more confident in our claims, but other claims such as “The results of this procedure may be able to represent that chia seeds taken as a dietary supplement have enough ALA to treat conditions such as cardiovascular disease and insulin resistance in humans” are not specifically addressed within this paper and serve only as statements for consideration in future studies and papers.

[1.13] The sentence was removed as suggested. [1.14] The original Scheme 1 was moved to Supporting Information as part of [M.3].

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[1.15] We appreciate the compliment included in the Minor Issues section. [1.16] Indent placed on the paragraph following Table 1.

[1.17] The reviewer writes “NMR spectra and interpretation should be moved to supporting information. Stick to the information that directly supports your conclusion for the main article.” Our paper aims to explore the chia seed as a proper venue for obtaining ALA. In order to do this, we need to be able to explain our methods of obtaining high yield. The characterization is necessary to do so, as the results and discussion largely discuss the characterization with regards to purity of the extraction process. [1.18] The wording has been changed to read, “Carboxyl groups are expected…”

[1.19] Indent placed on the paragraph following Figure 2. [1.20] In the sentence, “…a decrease in TG and LCA-CoA was recorded, to a value almost equal…” the comma following “recorded” was removed. [1.21] Indent placed on the paragraph following Table 2.

[1.22] The word “ones” was replaced with “nutraceuticals” as suggested. [1.23] Indent placed on the paragraph following Table 3.

[1.24] Again, thank you for the additional compliment. [1.25] Covered in [1.4].

[1.26] The beginning sentence of our conclusion was edited to both remove the phrase “our hypothesis was proven correct” and to restate our original hypothesis. It now reads, “It is concluded that the data supports our hypothesis that chia seeds taken as a supplement have the concentration of ALA necessary to reduce the effects of a high sucrose diet in rats.” [1.27] The phrase “as indicated by past literature” was removed from the sentence. The literature in question is already provided in the appropriate places. [1.28] Our original thought was that several, perhaps too many, previous manuscripts were using dotted lines and that we would try something different, but found that it is simply easier for the reader to understand if these lines are included.

Response to Reviewer 2

[2.1] The discussion on cost, as well as the analysis of the pulled data on the prices of other seeds known to contain ALA, is sufficient to answer your question. ALA sources come from seeds of several plants, such as the ones we listed. Looking up ALA in the Seed Oil Fatty Acid database explains this fact. ALA is not synthesized, it is extracted.

[2.2] See response [1.17].

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[2.3] This is not relevant to this study. For more information on this see the research done by Menendez. PMID: 15138577

[2.4] The hyperlink in question was removed from the reference. However, the URL is required for web page sources in ACS format.

Response to Reviewer 3

[3.1] Figures on the title page refer to our graphical abstract. They are necessary according to ACS format.

[3.2] The name of the compound was added to the graphical abstract. However, according to ACS guidelines, a caption is not the correct format for the graphical abstract.

[3.3] The paper was edited to change all instances of alpha linolenic acid to “ALA” after the first instance of using the compound name.

[3.4] The grammatical errors in the abstract were fixed. [3.5] Fixing the grammatical errors in the abstract fixes the readability of the abstract, adding to the effect of drawing the reader in. ALA was added to the abstract, in order to explain that it comes from chia seeds.

[3.6] A scheme of ALA was provided as of [M.2]. [3.7] The sentence was deleted as suggested, and the rest of the section was edited to reflect a more scholarly presentation. The term “best” was changed to “most effective”. [3.8] The term “cons” was replaced with “weaknesses” to further clarify.

[3.9] The extraction figure was moved, see [M.3]. [3.10] “The rats in the procedure were fed with a sucrose-rich diet for 3 months. Then, half remained on this diet while the other half was substituted an isocaloric amount of chia seeds in their diet. The control group was fed corn starch rather than sucrose.” was added to provide details of the control group in the materials and methods. [3.11] Added the citation as suggested by the reviewer.

[3.12] Thank you for the suggestion. The introduction and conclusion reflect ideas based off of this paragraph.

[3.13] See the response provided in [1.17]. [3.14] We believe an advanced discussion of the characterization is key to our ability to perform an accurate and reproducible experiment. For this reason, we disagree with your statement that it is unnecessary to go in depth about the characterization and purity.

[3.15] This edit is covered in [M.4]. [3.16] The following grammatical errors were fixed: “to the level of” and “can be seen as being” were removed.

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[3.17] Removed “The chia seed was shown to regulate the enzyme, which is important to rat metabolism.” from the first paragraph of the conclusion in order to affirm that supposition is found only in the second paragraph. [3.18] Edited as suggested.

We believe that our revisions have properly addressed the comments and concerns raised by the reviewers and that these revisions have notably improved our paper. Once again we would like to thank them for their effort in analyzing our work to make it more suitable for publication, which we hope you will now consider.

Best Regards, Ethan Baxter and Nathan Smith

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Alpha-linolenic acid in dietary chia seeds (Salvia hispanica) reduce effects of high sucrose diets

Ethan Baxter and Nathan Smith

Department of Chemistry, University of Missouri, Columbia, Missouri 65211

 

 

Alpha-Linolenic Acid  

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Abstract: The effect are reported of using chia seed as a dietary supplement for rats

whose metabolite levels and enzymatic activities in skeletal muscle have been

compromised due to ingesting sucrose-rich diets. Chia seeds are digested by the body to

produce alpha-linolenic acid, an essential omega-3 fatty acid. The rats were fed with a

sucrose-rich diet for 3 months. Then, half of the cohort remained on this diet while the

other half was substituted an isocaloric amount of chia seeds in their diet. The control

group was fed corn starch rather than sucrose. The chia seed group resulted in recovered

activities of lipogenic enzymes and skeletal muscle enzymes, as well as producing proper

levels of the respective enzyme metabolites, lipids and free fatty acids. Dietary

supplementation by chia seeds was able to reverse the induced effects of insulin

resistance and dyslipidemia.

 

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Introduction

Nutraceuticals are fortified foods or dietary supplements that provide health

benefits in addition to their basic nutritional value.1 Nutraceuticals contain substances

mostly attributed to natural food products, rather than synthetically formed compounds,

and are often used to prevent and treat diseases. Nutraceuticals as dietary supplements

have a health benefit, nutrition content benefit, function benefit, or some combination of

the three. Dietary supplements are produced to provide these benefits to individuals who

are lacking in overall health due to diet or certain genetic conditions, including

hypertension, coronary artery disease, and diabetes.

Unsaturated fatty acids are one specific type of nutraceutical. n-3 and n-6 fatty

acids are essential fatty acids that provide the means for important bodily functions.2

However, in some instances polyunsaturated fatty acids are prone to rapid oxidation,2

thereby losing their chemical structure and their function. Substances with high

concentrations of fatty acids should overcome the boundaries set by oxidation. One

particular type of polyunsaturated fatty acid is alpha-linolenic acid (ALA). ALA (C18:3)

is an n-3 fatty acid, as shown in Scheme 1, found in plants that has been shown to reduce

the risk of cardiovascular disease, reduce plasma lipid levels, and increase peripheral

insulin sensitivity in rats.3 ALA has also been connected to the treatment of individuals

with cardiovascular risks due to primary dyslipidemia4 – an abnormal amount of lipids in

the blood – and to the treatment of those with insulin resistance.5 This particular fatty

acid can be found in the seed of the chia plant, or Salvia hispanica. The chia seed has the

largest recorded concentration of ALA from sources of plant oils.3

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Scheme 1. Structure of Alpha-Linolenic Acid

Here we report whether feeding chia seeds to rats taking a sucrose-rich diet

produces any effect. We hypothesize that the ALA found in high concentration in chia

seeds will be able to resist oxidation and treat the dyslipidemia and insulin resistance

effects presented in one with a diet high in sucrose. The results of this procedure may be

able to represent that chia seeds taken as a dietary supplement have enough ALA to treat

conditions such as cardiovascular disease and insulin resistance in humans. This would

reduce the need to take ALA as a medication or a dietary pill to treat the same conditions.

Materials and Methods

The most effective way of extracting a pure sample of the oil from chia seeds

comes from supercritical fluid extraction (SFE), involving the use of water and

supercritical CO2.6 Extraction can also be performed using the solvent method or seed

compression, which respectively have weaknesses of loss of antioxidant content and

lower yield. SFE is optimally accomplished at a pressure of 408 atm and 80°C7,

producing the highest purity in the SFE and the highest ALA/LA content of the final

products. Oil yield can be increased further with pressure enhancement – but at the cost

of more time necessary to complete the procedure.7 A more detailed description of the

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extraction, including spectroscopic characterization and the extraction apparatus, can be

found in the supporting information.

The effectiveness of the chia seed as a dietary supplement can be evaluated based

on its ability to affect the molarity of specific metabolites in the gastrocnemius muscle, as

well as a change in enzymatic activities in the muscle. The three types of supplements

tested were a control group (CD), a sucrose-rich diet (SRD), and a sucrose- and chia-

seed-rich diet (SRD+Chia). The rats in the procedure were fed with a sucrose-rich diet for

3 months. Then, half remained on this diet while the other half was substituted an

isocaloric amount of chia seeds in their diet. The control group was fed corn starch rather

than sucrose. The metabolites tested were Triglycerides (TG), Long-chain acyl Coa

(LCA-CoA), Diacylglycerol (DAG), Glycogen, and Glucose-6-phosphate (G-6-P). The

enzymes tested were Hexokinase, activated Pyruvate Dehydrogenase Complex (PDHa),

and Glycogen Synthase (GS).3

The metabolites were measured using the euglycemic-hyperinsulinemic clamp

technique as performed by Ali and Yeap. Rats from each group were given an infusion of

highly purified porcine neutral insulin at about .8 units over 2 hours. In order to prevent

glycemia at this level of insulin input, synthetic glucose also was infused at a variable

rate dependent on the level of insulin. At the end of 2 hours, the gastrocnemius muscle

was quickly removed, where tests of TG, glycogen, G-6-P, GS, LCA-CoA, DAG,

hexokinase, and PDHc were completed to analyze concentrations of the metabolites and

activities of the enzymes.3

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Results and Discussion

The extraction of ALA is shown in Scheme S1 in the supporting information. The

chia seed, also known as Salvia Hispanica, was chosen because it has the highest

concentration of ALA that can be extracted out of any other plant. Two other acceptable

choices that could be substituted for chia seeds are kiwifruit seeds (Actinidia chinensis)

and shiso (Perilla frutescens), as they also contain very high concentrations of ALA.8

To ensure that our extraction had a high yield and was correctly executed, ALA

was characterized using 1H NMR and 13C NMR; these results are shown in Figures 1 and

2, respectively.

Figure 1. 1H NMR of alpha-linolenic acid

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The results of the 1H NMR are as expected based on the structure of the product,

indicating that the extraction was a success. Carboxyl groups are expected in the range of

10.5 – 12 ppm, which follows the absorption at 10.9.9 Vinylic hydrogens are expected in

the range of 4.6-5.9, as shown by the large absorption area from 5.54 to 5.17 ppm. The

rest of the absorptions are characteristic of hydrogens on secondary and tertiary groups,

while some of them are shifted downfield, due to the electron withdrawing capabilities of

the carboxylic group as well as interactions with the vinylic groups.

The results of the 13C NMR also indicate that the extraction product gave high

yield and was carried out correctly. One carbon appears at around 180 ppm. This carbon

is shifted far downfield as is typical for the carbon in the carboxylic group. Six of the

carbons show absorptions between 131 and 127 ppm. Alkene carbons show absorptions

in the 115-140 ppm range, which signifies that the compound has alkene groups. Alkyl

absorptions occur in the 0-50 ppm range. The shifts show the various CH2 and CH3

groups. There are 7 distinct peaks visible, while 4 more carbons are grouped up inside the

7 distinct absorptions. This can be explained by the chemical shifts caused by the vinylic

groups as well as the influence by surrounding CH2 groups.9

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Figure 2. 13C NMR of alpha-linolenic acid

The compound was further characterized as shown by complementary

characterization spectra that are available in the appendix. These additional spectra serve

as extra confirmation that the extraction mechanism was successful.

After successful completion of the euglycemic-hyperinsulinemic clamp technique,

which involved rats from three test groups given different dietary supplements, chemical

analysis of the gastrocnemius muscle was completed, as shown in Table 1.3

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Table 1. Metabolites and enzyme activities in gastrocnemius muscle of rats.

Diets CD SRD SRD+Chia

Metabolites Triglyceride (µmol/g wet tissue) 3.4 ± 0.1 6.7 ± 0.3 3.5 ± 0.2 Long-chain acyl CoA (nmol/g wet tissue) 6.2 ± 0.4 12.8 ± 0.8 5.6 ± 0.4 Diacylglycerol (nmol/g wet tissue) 108.3 ± 13.2 184.0 ± 8.9 135.5 ± 4.0 Glycogen (µmol/g wet tissue) 21.8 ± 1.7 22.3 ± 0.8 19.4 ± 0.4 Glucose-6-phosphate (µmol/g wet tissue) 0.42 ± 0.02 0.43 ± 0.03 0.39 ± 0.01

Enzyme activities

Hexokinase (pkat/mg protein) 746.0 ± 39.7 561.8 ± 23.3 656.8 ± 35.0 Glycogen synthase (% fractional activity) 36.1 ± 3.1 35.3 ± 2.9 33.8 ± 3.6 PDHa (% of total PdHc) 33.9 ± 0.7 21.0 ± 1.6 37.3 ± 1.0

The data found that the chia seed was able to cleanse chronically fed SRD rats of

excess metabolites, as well as help regulate enzymatic activities. The gastrocnemius

muscle of SRD-fed rats showed a significant increase in the metabolite concentrations of

TG, LCA-CoA and DAG, without significant changes in glycogen and G-6-P levels as

shown in CD-fed rats. The data shows that the chia seed-fed group (SRD+Chia) had the

ability to decrease metabolites to the level of values found in CD-fed rats. Specifically, a

decrease in TG and LCA-CoA was recorded to a value almost equal to that of the CD-fed

groups.

The euglycemic-hyperinsulinemic clamp technique was also utilized in the

analysis of adipose tissue of rats fed the CD, SRD, and SRD+Chia. This new data is

shown below in Table 2.3

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Table 2. Lipogenic enzyme activities in adipose tissue of rats.

Diets CD SRD SRD+Chia

Fatty acid synthase (pkat/mg protein) 246.7 ± 17.0 300.7 ± 14.0 239.0 ± 10.2 Glucose-6-phosphate dehydrogenase (pkat/mg) 509.3 ± 30.0 823.5 ± 72.2 555.1 ± 40.8 Acetyl-CoA carboxylase (pkat/mg protein) 480.1 ± 51.7 441.7 ± 27.2 442.0 ± 29.5 Malic enzyme (nkat/total fat weight) 23.0 ± 1.8 44.5 ± 2.8 31.8 ± 1.2

The activities of enzymes involved in lipogenesis increased in the SRD-fed group

as compared to the CD-fed group. However, when the dietary chia seed was added to the

SRD, a significant reduction of FAS and G-6-PDH activities was observed, where they

reached values similar to what was initially recorded for the CD-fed group. Malic enzyme

was decreased, but not to the level of the CD.

Up to this point, it has been tested that Chia seeds have been shown to be an

excellence source of omega-3-fatty acids, helping regulate metabolism in rats. When

comparing the cost of manufacturing this nutraceutical with comparable nutraceuticals, it

can be seen based on the cost of the starting materials that the chia seed provides the most

effective outlet for an organic source of ALA (Table 3).10 The price of the chia seed is

much less in comparison to the perilla seed and the flax seed, being 15% the price of the

perilla seed and 49% the price of the flax seed.

Table 3. Prices of comparable nutraceuticals.

Plant   Linnaean  name   %  ALA  of  oil   Price  (USD/50g)  Chia   Salvia  hispanica   64   2.36  Perilla   Perilla  frutescens   58   15.28  Flax   Linum  usitatissimum   55   4.8125  

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Furthermore, the chia seed contains a higher percent of ALA in the oil. The chia

seed oil is collected through the extraction described. The ALA is the key component of

the nutraceutical: the part that is actively playing a role in metabolism.

Finally, tests were performed on the concentration of enzymes after adding the

dietary chia seed. In order to do this, immunoblotting of the gastrocnemius muscle was

required, as the thickness of the bands will show the relative concentrations of the

enzymes in the muscle. In this particular experiment, the membrane concentration of

Protein Kinase C Theta (PKCθ) was compared within the CD, SRD, and SRD+Chia

groups as shown in Figure 3 below.

Figure 3. Concentrations of PKCθ in the CD, SRD, and SRD+Chia groups

The SRD group shows almost two times the concentration of the enzyme.3 This is

characteristic of the SRD group, which has shown elevated metabolites as a result of an

increased sugar intake. Contrary to this, the chia seed can be seen on this graph working

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its primary function: maintaining a normal and healthy level of metabolites and enzymes.

This is utilized by switching the source of fat away from the sucrose-rich corn oil diet to

the chia seed containing the omega-3 fatty acid ALA as its main source of fat, which is

much healthier and easier to maintain by the body.

Conclusion

It is concluded that the data supports our hypothesis that chia seeds taken as a

supplement have the concentration of ALA necessary to reduce the effects of a high

sucrose diet in rats. The extraction of chia seeds produced a high yield product, at little

cost of starting material. 1H NMR and 13C NMR characterization (shown in results) as

well as supplemental characterization shown in the appendix show that the chia seed

extraction produced the desired ALA product successfully. Supercritical Fluid Extraction

was proven as a viable source of high yield ALA. The hypothesis regarding the function

of the nutraceutical was supported through the testing of the three groups of rats. The

resulting metabolite and enzyme concentrations were normalized in the gastrocnemius

muscle of the rat in the SRD+Chia groups when they were compared to the SRD group.

The concentrations of the metabolites in the wet tissue of the muscle, in particular the TG

and DAG, were shown to normalize when supplemented with the dietary chia seed.

Enzyme activity was affected when the concentration PKCθ in the SRD group was

almost twice the concentration of the SRD+Chia group.

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The data presented demonstrates that a diet rich in chia seeds will improve the

activities of enzymes involved with lipid and glucose metabolism that have been

impaired by a high sucrose diet in rats. The high levels of triglyceride, long-chain acyl

CoA, and diacylglycerol and high activities of G6PD and malic enzyme found in SRD

have been reduced in SRD+Chia. This restoration in normal enzymatic function has

effectively reduced the insulin resistance and the hyperlipidemia found in skeletal

muscle. Taking chia seeds as a dietary supplement may therefore have a similar effect in

humans, though further experimentation will be necessary to confirm. These further tests

will determine whether ALA is effective in humans to reduce cardiovascular disease and

insulin resistance, and whether dietary chia seeds are an effective source of ALA.

Supplemental Material Available: The appendix contains a more detailed

description of extracting alpha-linolenic acid through supercritical fluid extraction.

Spectroscopy images on alpha-linolenic acid are also contained in this section. The

appendix is found directly following this paper.

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References

(1) Nutraceutical. Merriam-Webster Dictionary. 2014.

(2) Jayasinghe, C.; Gotoh, N.; Wada, S. Food Chem. 2013, 141, 3077-3084.

(3) Oliva, M. E.; Ferreira, M. R. Prostaglandins Leukotrienes and Essential Fatty Acids,

2013, 89, 279-289.

(4) Sala-Vila, A.; Cofán, M.; Núñez, I.; Gilabert, R.; Junyent, M.; Ros, E. Atherosclerosis

2011, 214, 209-214.

(5) Muramatsu, T.; et al. Preventive Medicine 2010, 50, 272-276.

(6) Oman, M. Macedonian J Chem and Chem E 2013, 32, 183-226.

(7) Ali, N; Yeap, S. J. Biomed. Biotechn. 2012, 1-9.

(8) Max Rubner-Institut. Seed Oil Fatty Acids. http://sofa.mri.bund.de/ (accessed April

15, 2014).

(9) Schoffstall, A. M.; Gaddis, B. A.; Druelinger, M. L. In Microscale and Miniscale

Organic Chemistry Laboratory Experiments; McGraw-Hill: Boston, 2003.

(10) My Seeds Company. http://www.myseeds.co/ (accessed April 15, 2014).

  1  

Supporting Information

Alpha-linolenic acid in dietary chia seeds (Salvia hispanica) reduce effects of high

sucrose diets

Ethan Baxter and Nathan Smith

Department of Chemistry, University of Missouri, Columbia, Missouri 65211

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Appendix Contents

Alpha-linolenic acid extraction

Process……………………………………………….3 Apparatus…………………………………………….4

Total chia seed fatty acid content…………………………....5

Alpha-linolenic acid spectroscopy

1H-NMR……………………………………………..6 13C-NMR…………………………………………….7 UV-Vis………………………………………………8

Bibliography…………………………………………………9

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Alpha-linolenic acid extraction

Chia seeds are the leading source of ALA, and are therefore the most appropriate

sources to extract this particular fatty acid. Extraction of ALA is performed using

supercritical fluid extraction with the apparatus shown in Scheme S1. The apparatus

performs in temperatures up to 400°C and pressures up to 690 bar, making the optimal

conditions of 80°C and 408 atm (413.4 bar) for ALA extraction feasible. Using

supercritical carbon dioxide for extraction allows for a greater separation of fatty acids.

With this apparatus, one can perform up to four extractions in parallel.

Prior to extraction, the chia seeds were cleaned, milled (MF 10 basic Microfine

grinder drive, IKA 5250), sieved, and stored in a 4°C refrigerator for fewer than 24 hours.

In each extraction column (EC), the chia seed flour is saturated with water and let sit. The

ends of the EC are plugged with glass wool, so as to not let solid escape. Through the

modifier inlet, distilled water is allowed to flow in to saturate the CO2 stream. CO2 is

pumped through the EC at a constant flow rate of 1.82 kg/h. This creates a pressure

gradient, which allows the separation and precipitation of the ALA through the

micrometering valve (MV) and into the glass vial (GV) for collection. Extraction was

performed for 4 hours.

According to a procedure by Ixtaina et al., the average content of ALA compared

to the total fatty acid content of chia seed extract is 54.38%. The total fatty acid

composition of chia seeds from this procedure is given in Table S1. The percent yield of

total oil extraction was not provided.

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Scheme S1. Extraction Outline of Alpha-Linolenic Acid

V – modifier inlet; EC – extraction column; MV – micrometrical valve; GV – glass vial; R – rotameter.

  5  

Table S1. % concentration of fatty acids from extraction of chia seeds with supercritical CO2. Run   C16:0   C18:0   C18:1   C18:2   C18:3  1   10.1   2.8   10.3   28.0   48.8  2   11.5   4.1   6.6   21.0   56.8  3   10.7   2.5   7.5   20.0   59.2  4   9.6   4.0   7.8   19.8   58.8  5   14.0   5.2   8.9   20.8   51.1  6   10.4   5.3   5.5   21.8   57.0  7   11.9   4.1   11.1   21.5   51.4  8   12.5   4.9   7.5   20.8   54.3  9   7.5   13.0   5.3   30.0   44.4  10   9.2   4.3   10.0   22.3   54.2  11   6.8   3.1   7.1   30.5   52.5  12   8.0   4.9   3.9   35.8   47.5  13   11.4   4.4   7.6   20.6   56.1  14   8.7   3.4   4.9   19.6   63.4  15   10.5   4.5   6.8   21.8   56.4  16   9.5   4.2   6.2   20.6   59.5  17   9.8   4.3   6.4   20.9   58.6  18   10.1   2.8   10.3   28.0   48.8  

C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid

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Alpha-linolenic acid spectroscopy

Figure S1. 1H NMR Spectra of Alpha-Linolenic Acid, CDCl3, 89.56 MHz. d : .973, 1.48 to 1.26, 1.64, 2.13 to 1.95, 2.33, 2.80, 5.54 to 5.17, 10.9.

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Figure S2. 13C NMR Spectra of Alpha-Linolenic Acid, CDCl3, 25.16 MHz. d: 14.30, 20.61, 24.72, 25.60, 25.68, 27.26, 29.13, 29.19, 29.63, 34.17, 127.19, 127.84, 128.30, 130.23, 131.94, 180.55.

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Figure S3. UV-Visible Spectra of Alpha-Linolenic Acid, λmax = 214.5

0  

0.2  

0.4  

0.6  

0.8  

1  

200   205   210   215   220   225  

Absorbance  

Wavelength  (nm)  

  9  

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