Antioxidants and Their Use in Stabilizing Lemon Oil

GINGER (Zingiber officinale):

ANTIOXIDANTS AND THEIR USE IN STABILIZING LEMON OIL

KATHRYN BARDSLEY

A dissertation submitted to the

Graduate School-New Brunswick

Rutgers, The State University of New Jersey

in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

Graduate Program in Food Science

written under the direction of

Dr. Chi-Tang Ho and Dr. Henryk Daun

and approved by

______

New Brunswick, New Jersey

May 2013

ABSTRACT OF THE DISSERTATION

Ginger (Zingiber officinale):

Antioxidants and Their Use in Stabilizing Lemon Oil

By KATHRYN BARDSLEY

Dissertation Directors: Dr. Chi-Tang Ho and Dr. Henryk Daun

Nine ginger powders from various parts of the world have been studied for their antioxidant strength by way of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC). Of those tested, it was found that the acetone extract of Nigerian ginger from Synthite had the greatest antioxidant power.

The extract was fractionated using the SepBox® and each fraction collected was tested for its ORAC value. The fractions with the greatest ORAC values are listed in increasing order, and were identified and confirmed by LC/MS/UV, MS/MS and

HRMS: [6]-gingerdiol, octahydrocurcumin, [6]-gingerol, [4]-gingerdiol and hexahydrocurcumin.

Select concentrations of each antioxidant were added to a single-fold

Argentinian lemon oil, which were then placed in a thermally accelerated storage chamber for four weeks. Upon removal, the lemon oils were analyzed by GC, measured for their peroxide values and tasted by an expert citrus panel in a high- acid tasting solution.

From the initial tasting, the two most preferred samples contained the antioxidants, tocopherols and tetrahydrocurcumin, which were also the two samples with the lowest peroxide values. Regarding the individual attributes, however, there weren’t significant sensory differences between the oils; therefore, the tasting solutions of these oils were placed in the chamber for an additional two weeks. The sequential tasting revealed that hexahydrocurcumin was the most effective antioxidant in preserving the flavor quality of the lemon beverage. These findings prove that incorporating antioxidants can improve the flavor and stability lemon oil.

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ACKNOWLEDGEMENT AND DEDICATION

I would like to acknowledge Rutgers University and International Flavors and Fragrances, Inc. (IFF) for supporting me through this entire endeavor, as well as, my advisors, Dr. Chi-Tang Ho and Dr. Henryk Daun, and my friends at IFF, Dr.

Michael Chen, Dr. Sonia Liu, Dr. Hou Wu and Michael Hughes, who readily helped me in their area of expertise. I’d also like to thank my family and friends who have been tremendously supportive throughout the most arduous days; thank you for your care.

When I think of how I got here, the reflection is a bit hazy, but when I think about who got me here, the path becomes quite clear.

Mom: You were my first great influence; always giving your entire self to make life easier for your children. Thank you for the past thirty years of unconditional love, but most of all, thank you for your encouragement to always follow the music of my heart.

Dad: You define resilience. Thank you for teaching me to work hard while reminding me to enjoy the moments in between.

TV: Thank you for inspiring me and making me believe that, what I doubted was possible for myself, was truly attainable. You are the true definition of a mentor with whom I am so thankful for.

Lastly, to my “life coach” and to whom I dedicate this work to, my husband,

Frank: I could not have done this without you... Your love let me soar.

TABLE OF CONTENTS

ABSTRACT OF THE DISSERTATION…………………………………………………………………… ii

ACKNOWLEDGEMENT AND DEDICATION………………………………………………………… iv

LIST OF TABLES …..……….…………………………………………………………………………….…...... x

LIST OF SCHEMES …………………………………………………………………………………………… xii

LIST OF FIGURES …………………………………………………………………………………………… xiii

LIST OF APPENDICES ………………………………………………………………………………..…… xiv

1. INTRODUCTION………………………………………………..…….………….………………………… 1

1.1. The History and Background of Ginger

1.2. Traditional Uses of Ginger and Its Uses Today

1.2.1. As a Spice and Flavorant

1.2.2. For Medicinal Purposes

1.3. The Chemical Components of Ginger

1.4. The Use of Ginger as an Antioxidant

1.4.1. Health Related Active Compounds

1.5. The Use of Antioxidants in Foods and Flavors

2. LITERATURE REVIEW…………………………………………………………………..……………… 7

2.1. Ginger as an Antioxidant

2.1.1. Antioxidant Studies of Ginger Extracts

2.1.2. Comparative Antioxidant Studies of Active Compounds Found in Ginger

2.2. Antioxidants in Food Applications

2.2.1. The Use of Ginger as an Antioxidant in Food

2.3. Citrus Instability

2.3.1. Limonene Oxidation

2.3.2. Citral Degradation

2.3.3. Stability Efforts

3. HYPOTHESIS ……………………………………………...…………………………...…………....…… 18

4. RESEARCH OBJECTIVES ………………………………………..…………………………...……… 19

4.1. Ginger Selection

4.2. Fractionation and ORAC Monitoring

4.3. Identification of Strongest Antioxidants

4.4. Use of Antioxidants in Lemon Oil

5. EXPERIMENTAL …………………………………………………………..………………………...….. 21

5.1. Materials

5.1.1. Ginger Powders

5.1.2. Solvents, Reagents and Supplies

5.2. Instruments and Equipment

5.3. Methods and Protocols

5.3.1. Moisture Content

5.3.2. Extraction of Ginger Powders and Fractionation of Synthite Nigerian Acetone Extract

5.3.2.1. Acetone Extracts Using Orbital Shaker

5.3.2.2. Hexane Extracts Using Speed Extractor

5.3.2.3. Percolator Extract for SepBox®

5.3.2.4. SepBox® Fractionation

5.3.3. Analytical Work on Ginger Extracts and Lemon Oils

5.3.3.1. Gas Chromatogram (GC) Work on Ginger Acetone Extracts

5.3.3.2. Gas Chromatogram/Mass Spectrometry (GC/MS) Work on Lemon Oils

5.3.3.3. High Performance Liquid Chromatography (HPLC) Profiling

5.3.3.4. Structure Identification of Major Components in Select Ginger Fractions

5.4. Antioxidant Assays

5.4.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Activity

5.4.2. Oxygen Radical Absorbance Capacity (ORAC)

5.4.3. Peroxide Value Assay

5.5. Syntheses of Ginger-related Compounds

5.5.1. [4]-Gingerol

5.5.2. [4]-Shogaol

5.5.3. [4]-Gingerdiol

5.5.4. [6]-Gingerdiol

5.5.5. Octahydrocurcumin

5.6. Sensory Evaluation of Lemon Oils

5.6.1. Sensory Evaluation of Lemon Oils after Thermal Acceleration

5.6.2. Sensory Evaluation of Lemon Beverages after Thermal Acceleration

5.7. Statistical Analysis of Lemon Oils and Peroxide Values

5.7.1. Statistical Analysis of Peroxide Values

5.7.2. Statistical Analysis of Tasting Results

6. RESULTS AND DISCUSSSION………………………………………………………………….…… 36

6.1. General Background Information

6.1.1. Growing Regions of Ginger Samples

6.1.2. Moisture Content of Ginger Powders

vii

6.1.3. Sensory Descriptions of Ginger Samples

6.1.3.1. Aroma of Dried Ginger Powders

6.1.3.2. Blotter Aroma of 10% Dilutions in Acetone

6.1.3.3. Tasting Comments of 0.1% Solutions in Water

6.1.4. Extraction of Ginger Powders

6.1.4.1. Acetone Extracts Using Orbital Shaker

6.1.4.2. Hexane Extracts Using Speed Extractor

6.1.5. Analytical Work: GC and HPLC

6.2. Ginger Selection

6.2.1. Determination of Gingerol and Shogaol Content

6.2.2. Antioxidant Activity Studies

6.2.2.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Activity

6.2.2.2. Oxygen Radial Absorbance Capacity (ORAC)

6.2.2.3. Summary of Hexane Extracts versus Acetone Extracts

6.3. Fractionation and ORAC Monitoring

6.3.1. Ginger Extract Fractionation via SepBox®

6.3.2. ORAC Assay of Each Fraction

6.3.3. Identification of Antioxidants

6.3.3.1. Proposed Active Compounds

6.3.3.2. Confirmed Active Compounds

6.4. Use of Antioxidants in Lemon Oil

6.4.1. Determination of Antioxidant Concentrations via ORAC

6.4.2. Antioxidants in Lemon Oil

viii

6.4.3. Peroxide Study of the Lemon Oils with and without Ginger-related Antioxidants

6.4.4. Sensory Validation of Ginger-related Antioxidants in Lemon Drink

6.4.4.1. Sensory Validation of Lemon Oils after Thermal Treatment

6.4.4.2. Sensory Validation of Lemon Beverages after Thermal Treatment

6.4.5. GC Analysis of Markers Representing Limonene Oxidation and Citral Degradation

7. CONCLUSION ……………..………………………………………………………………………………. 76

8. FUTURE PERSPECTIVES ……………………………………………………………………………. 78

9. REFERENCES……………………………………………………………………...………………………. 79

10. APPENDIX……………………………………………………………………………………….………… 83

LIST OF TABLES

Table 1. Average moisture content of ginger powders

Table 2. Yields of acetone extracts using orbital shaker

Table 3. Yields of hexane extracts using speed extractor

Table 4. Ranking of gingerol and shogaol summation

Table 5. DPPH scavenging activity of ginger extracts

Table 6. ORAC values for hexane and acetone extracts at 0.01%

Table 7. Compilation of hexane extracts: values and rankings

Table 8. Compilation of acetone extracts: values and rankings

Table 9. Synthite Nigerian SepBox® fractions with the highest ORAC values

Table 10. ORAC values for known ginger antioxidants

Table 11. Active compounds identified from Synthite Nigerian ginger fractions

Table 12. Levels of antioxidants added to Argentinian lemon oil

Table 13. Mean peroxide values of Argentinian lemon oils

Table 14. Data analysis of peroxide values from first set of lemon oils

Table 15. Data analysis of peroxide values from second set of lemon oils

Table 16. Paired comparison of significance between sets of lemon oils

Table 17. Lemon beverage descriptors

Table 18. Statistical analysis of lemon beverage tastings for the attribute, peely

Table 19. Statistical analysis of lemon beverage tastings for the attribute, citral

Table 20. Statistical analysis of lemon beverage tastings for the attribute, candied

Table 21. Statistical analysis of lemon beverage tastings for the attribute, juicy

Table 22. Statistical analysis of lemon beverage tastings for the attribute, oxidized

Table 23. Statistical analysis of lemon beverage tastings for the attribute, cooked

Table 24. Statistical analysis of lemon beverage tastings for the attribute, plastic/phenolic

Table 25. Statistical analysis of lemon beverage tastings for the attribute, cherry/ medicinal

Table 26. Statistical analysis of lemon beverage tastings for the attribute, piney

Table 27. Statistical analysis of lemon beverage tastings for the attribute, soapy/ green

Table 28. Statistical analysis of lemon beverage tastings for the attribute, turpentine

Table 29. Degradation markers for GC/MS analysis of lemon oils

Table 30. Peak areas of degradation markers of tasted lemon oils

LIST OF SCHEMES

Scheme 1. Proposed mechanism for the formation of p-cresol from citral

Scheme 2. The reduction of DPPH

xii

LIST OF FIGURES

Figure 1. S-[6]-gingerol

Figure 2. [6]-shogaol

Figure 3. Isolated compounds from ginger

Figure 4. D-limonene

Figure 5. Reaction products of limonene

Figure 6. Structures (a-i) of the identified compounds:

Figure 6a. F126/ F111: Hexahydrocurcumin (5-hydroxy-1,7-bis(4-hydroxy-3- methoxyphenyl)- 3-heptanone)

Figure 6b. F142: [4]-Gingerdiol ((3R,5S)- 1-(4-hydroxy-3-methoxyphenyl)-3,5- octanediol)

Figure 6c. F167: [6]-Gingerol ((5S)- 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3- decanone)

Figure 6d. F110-1: 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3- methoxyphenyl)-3,5-heptanediol

Figure 6e. F110-2: Octahydrocurcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5- heptanediol)

Figure 6f. F165-1: [6]-Gingerdiol ((3S,5R)- 1-(4-hydroxy-3-methoxyphenyl)-3,5- decanediol)

Figure 6g. F165-2: [6]-Gingerol ((5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)- 3-decanone)

Figure 6h. F108-1: 1-(3,4-dihydroxy-5-methoxyphenyl)-5-hydroxy-7-(4-hydroxy-3- methoxyphenyl)-3-heptanone)

Figure 6i. F108-2: 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-(4-hydroxy-3- methoxyphenyl)-3-heptanone

Figure 7. Citral ratings of lemon oils after storage

Figure 8. Flavor attributes of most preferred antioxidants in lemon beverages

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LIST OF APPENDICES

Appendix 1: Chemical Composition of Ginger Oleoresins by GC (Alphabetical)

Appendix 2: Chemical Composition of Ginger Oleoresins by GC (Retention Time)

Appendix 3. GC of Acetone Extract Synthite Rajakumari Ginger

Appendix 4. GC of Acetone Extract Synthite Irutti Ginger

Appendix 5. GC of Acetone Extract Synthite Nigerian Ginger

Appendix 6. GC of Acetone Extract Synthite Shimoga Ginger

Appendix 7. GC of Acetone Extract Whole Herb Nigerian Ginger

Appendix 8. GC of Acetone Extract Whole Herb Indian Ginger

Appendix 9. GC of Acetone Extract Buderim Australian Ginger

Appendix 10. GC of Acetone Extract Buderim Fijian Ginger

Appendix 11. GC of Acetone Extract PharmaChem Chinese Ginger

Appendix 12. HPLC of Hexane Extract Synthite Rajakumari Ginger

Appendix 13. HPLC of Hexane Extract Synthite Irutti Ginger

Appendix 14. HPLC of Hexane Extract Synthite Nigerian Ginger

Appendix 15. HPLC of Hexane Extract Synthite Shimoga Ginger

Appendix 16. HPLC of Hexane Extract Whole Herb Nigerian Ginger

Appendix 17. HPLC of Hexane Extract Whole Herb Indian Ginger

Appendix 18. HPLC of Hexane Extract Buderim Australian Ginger

Appendix 19. HPLC of Hexane Extract Buderim Fijian Ginger

Appendix 20. HPLC of Hexane Extract PharmaChem Chinese Ginger

Appendix 21. HPLC of Acetone Extract Synthite Rajakumari Ginger

Appendix 22. HPLC of Acetone Extract Synthite Irutti Ginger

xiv

Appendix 23a. HPLC of Acetone Extract Synthite Nigerian Ginger

Appendix 23b. HPLC of Hexane Washed, Acetone Extract Synthite Nigerian Ginger

Appendix 24. HPLC of Acetone Extract Synthite Shimoga Ginger

Appendix 25. HPLC of Acetone Extract Whole Herb Nigerian Ginger

Appendix 26. HPLC of Acetone Extract Whole Herb Indian Ginger

Appendix 27. HPLC of Acetone Extract Buderim Australian Ginger

Appendix 28. HPLC of Acetone Extract Buderim Fijian Ginger

Appendix 29. HPLC of Acetone Extract PharmaChem Chinese Ginger

Appendix 30: Peak Area Summation of Gingerols and Shogaols from HPLC

Appendix 31. Hexane Fluorescence Decay Curve

Appendix 32. Acetone Fluorescence Decay Curve

Appendix 33. The LC/MS Chromatograms of F166 and F167

Appendix 34. The LC/MS Chromatograms of F126, F111, F142 and F167

Appendix 35. The LC/MS Chromatograms of F108, F110 and F165

Appendix 36. The LC/MS and MS2 of F126 and the Commercial Standard (Hexahydrocurcumin) for Confirmation

Appendix 37. 1H-NMR of Hexahydrocurcumin

Appendix 38. The LC/MS and MS2 of F167 and the Commercial Standard ([6]- Gingerol) for Confirmation

Appendix 39. 1H-NMR of [6]-Gingerol

Appendix 40. The LC/MS and MS2 of F142 and the Synthetic Standard ([4]- Gingerdiol) for Confirmation

Appendix 41. 1H-NMR of [4]-Gingerdiol

Appendix 42. The LC/MS and MS2 of F110 and the Synthetic Standard (Octahydrocurcumin) for Confirmation

Appendix 43. 1H-NMR of Octahydrocurcumin Pure Fraction 5A

Appendix 44. The LC/MS and MS2 of F165 and the Synthetic Standard ([6]- Gingerdiol) for Confirmation

Appendix 45. 1H-NMR of [6]-Gingerdiol

Appendix 46. Optimizing Antioxidant Concentrations Using ORAC Assay

Appendix 47. LCMS of Tetrahydrocurcumin

Appendix 48. 1H-NMR of [4]-Gingerol

Appendix 49. 1H-NMR of [4]-Shogaol

Appendix 50. Data Related to First Set of Antioxidants in Lemon Oil

Appendix 51. Data Related to Second Set of Antioxidants in Lemon Oil

Appendix 52. Bar Chart to Display Significance of First Set of Lemon Oils

Appendix 53. Bar Chart to Display Significance of Second Set of Lemon Oils

Appendix 54. Bar Chart to Display Significance of Paired Comparisons

Appendix 55. Peak Areas from GC of Degradation Markers for Each Lemon Oil

Appendix 56. GC of Original Starting Argentinian Lemon Oil

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1. INTRODUCTION

1.1. The History and Background of Ginger

Ginger, botanically known as Zingiber officinale Roscoe, belongs to the

Zingiberaceae family, which encompasses 47 genera and 1400 species, including turmeric (Curcuma longa) and cardamom (two main genera, Elettaria and Amomum.)

The genus, Zingiber, contains 150 species; however, the only species extensively used for flavoring is Z. officinale (Ravindran and Nirmal Babu, 2005). It is grown from

April to December at an optimal elevation between 300 and 900m (Pruthy, J.S.,

1993); requiring a warm, humid climate while preferring light shade (Jayachandran et al., 1991).

Ginger has been cultivated in southern Asian countries for over 3000 years and its discovery and value as a spice and medicinal plant has been well documented.

Ginger has been mentioned in several places throughout history: “Round amongst them (the righteous in paradise) is passed vessels of silver and goblets made of glass… a cup, the admixture of which is ginger” (Koran 76: 15-17). One of the earliest references made was by Rabbi Benjamin Tudella from his travels between 1159 and

1173 A.D. who described the cultivation and trade of spices coming from the ancient port of Quilon, in the State of Kerala (Mahindru, 1982). The most significant event that changed the history of the spice trade was the landing of Vasco da Gama in

1498 on the west coast of India, Malabar Coast (Kerala) (Mahindru, 1982).

Additional documentation dating back to 1298 A.D. was found in Marco Polo’s travelogue stating that “good ginger grows here and is known by the name of Quilon ginger” (translation by Menon, 1929).

Since its discovery, India has been the largest producing country of ginger.

Together, two of the states within the country, Kerala and Meghalaya, make up 30 to

40% of the world’s total ginger production (FAOSTAT Database). The second largest ginger producer is Nigeria, followed by several other producers and exporters dispersed throughout the world: China, Jamaica, Taiwan, Sierra Leone, Fiji,

Mauritius, Indonesia, Brazil, Costa Rica, Ghana, Japan, Malaysia, Bangladesh,

Philippines, Sri Lanka, Solomon Islands, Thailand, Trinidad and Tobago, Uganda,

Hawaii, Guatemala and many Pacific Ocean Islands (Ravindran and Nirmal Babu,

2005).

1.2. Traditional Uses of Ginger and Its Uses Today

1.2.1. As a Spice and Flavorant

Ginger is an ingredient found in the world’s cuisine. Legend has it that the first gingerbread was made by a baker on the Isle of Rhodes near Greece around

2400 B.C. In the 1500’s, gingerbread was known to be Queen Elizabeth I’s favorite treat (Farrell, 1985) and during the Middle Ages, tavern keepers would keep a constant supply of ground ginger powder so customers could sprinkle it on their beer

(Rosengarten, 1969). Today it is used in several products including Indian masala mixes, pumpkin pie spice, ginger ale, etc. Ginger based products are less popular in the Western world as compared to Australia, Thailand, Japan and China; the Buderim

Company in Queensland, Australia, for example, produces more than 100 ginger- based products (Ravindran and Nirmal Babu, 2005).

1.2.2. For Medicinal Purposes

Ginger has been used for medicinal purposes long before its understanding.

Traditionally ginger is used in both fresh and dried forms in Chinese, Indian,

Indonesian and Japanese medicines for the treatment of: arthritis, rheumatism, sprains, muscular aches, asthma, sore throats, motion sickness, indigestion, nausea, vomiting, diarrhea, constipation, hypertension, dementia, etc. (Cho et al., 2001;

Badreldin et al., 2008; Pharmacopoeia of the People’s Republic of China, 2010). In ancient India, ginger was primarily used as a medicine rather than as a flavorant and was referred to as the mahaoushadha (the great medicine) and vishwabheshaja (the universal cure) (Ravindran and Nirmal Babu, 2005). Ayurveda is a traditional approach to medicine that is native to India; ginger has been widely used in

Ayurvedic medicine to treat a variety of gastrointestinal ailments. Modern homeopathic uses of ginger are quite similar and are popularly used for the treatment of indigestion, nausea due to motion sickness, pregnancy and for patients undergoing chemotherapy.

1.3. The Chemical Components of Ginger

Steam distillation and supercritical carbon dioxide (CO2) extraction yield an essential oil containing volatile components, whereas, solvent extraction yields oleoresins containing non-volatiles and tastants (Ravindran and Nirmal Babu, 2005).

The first chemical study of ginger (Cochin) was done by J.O. Thresh in 1879

(Yearbook of Pharmacy, 1879, 1881 and 1882).

Some of the main volatiles identified by Connell in 1970 are “the

sesquiterpene hydrocarbons: (-)- -zingiberene, (+)-ar-curcumene, -bisabolene, - sesquiphellandrene, farnesene, -selinene, -elemene and -zingiberene. Other monoterpene hydrocarbons identified are: -pinene, -pinene, myrcene, - phellandrene, limonene, para-cymene, cumene, and oxygenated compounds: 1,8- cineole, d-borneol, linalool, neral, geranial, bornyl acetate; aliphatic aldehydes: nonanal, decanal; ketones: methylheptenone; alcohols: 2-heptanol, 2-nonanol; esters of acetic and caprylic acid and chavicol” (Connell, 1970).

The non-volatiles known to be responsible for the pungency of ginger are the gingerols and shogaols. [6]-gingerol was first identified by Lapworth in 1917; in

1969, Connell and Sutherland established the S-configuration for the hydroxyl group

(Figure 1). Gingerols undergo dehydration readily due to the thermally labile beta- hydroxy-keto group, thereby forming the corresponding shogaols (Figure 2).

O OH

HO O

Figure 1. S-[6]-gingerol

HO O

Figure 2. [6]-shogaol

The production of n-hexanal after alkaline hydrolysis of gingerol afforded the name [6]-gingerol while the name shogaol, was derived from the Japanese word for ginger, “shoga”. Gingerols and shogaols are not only responsible for the pungency of the ginger oleoresin but have been proven to be responsible for its antioxidant capability as well (Kikuzaki and Nakatani, 1993).

1.4. The Use of Ginger as an Antioxidant

1.4.1. Health Related Active Compounds

Antioxidants are present in nutraceuticals which refers to foods that have inherent health benefits greater than their dietary need and are consumed to help treat or prevent specific diseases. Nutraceuticals are typically plants, fruits, vegetables, roots and seeds because they internally produce their own antioxidants to combat oxidative stress offering a source of natural antioxidants. Carotenoids, flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols, tocotrienols are some of the natural antioxidants produced by the before mentioned botanicals for their own oxidative protection (Ghasemzadeh et al., 2010).

Oxidative damage in living tissue results in an inflammatory response which can also lead to the increased risk of chronic diseases such as cancer, coronary atherosclerosis and other age-related, degenerative diseases (Stoilova et al., 2007;

Astley, 2003). Dietary antioxidants found in nutraceuticals help to eliminate or prevent the accumulation of the damaging oxidative products within the botanical and have proven to be useful for human health as well.

Gingerols and shogaols are the most well-known and studied antioxidants in

ginger and have shown to have several pharmacological effects including the inhibition of prostaglandin biosynthesis, anti-hepatotoxicity, cardiotonic and anti- platelet (Cho et al., 2001).

1.5. The Use of Antioxidants in Foods and Flavors

Similarly to the auto-oxidation of lipids in biological membranes, oxidation degradation can also occur in food. Fat oxidation, for example, leads to an occurrence of several chain reactions forming double bonds, alcohols, aldehydes and ketones which generate off-flavors and the reduce the nutritional value (Stoilova et al., 2007).

For decades, food technologists and flavorists around the world have used antioxidants to retard or inhibit the spoilage and rancidity of foods caused by oxidation reactions. The most common synthetic antioxidants used in the food and flavor industry are butylated hydroxytoluene (BHT) and butylated hydroxyanisole

(BHA). However, as the trend amongst food consumers and suppliers to avoid synthetic additives continues to increase, so does the importance of conducting research that identifies, understands and utilizes antioxidants of natural origin.

The efficiency of ginger extracts, and that of gingerols and shogaols, has been compared to the synthetic antioxidants, BHT and BHA, and select natural antioxidants, namely, -tocopherol and ascorbic acid, which will be discussed in the literature review section of this composition.

2. LITERATURE REVIEW

2.1. Ginger as an Antioxidant

2.1.1. Antioxidant Studies of Ginger Extracts

The efficiency of ginger extracts have been compared to that of the synthetic antioxidants, BHT and BHA, and the natural antioxidant, -tocopherol. For example, the acetone extracts of the rhizomes of two ginger species, Z. cassumunar and Z. officinale, were proven to be comparable and in some cases, stronger antioxidants in the inhibition of lipid peroxidation by ferric thiocyanate (FTC) and thiobarbituric acid reactive species (TBARS) methods. In this study, Z. officinale was a stronger antioxidant than -tocopherol and comparable to that of BHT (Jitoe et al., 1992;

Kikuzaki and Nakatani, 1993).

CO2 ginger extracts, which are rich in polyphenols, are known to donate their hydrogen atoms to reduce free radicals. In a study by Stoilova et al. (2007), the efficacy of the CO2 extract of Z. officinale was compared to BHT by measuring the ability to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and inhibit the oxidation of linoleic acid. The ginger extract had a significantly higher scavenging ability regarding DPPH and in the study of lipid oxidation, the ginger extract was better at both, inhibiting the formation of conjugated dienes, as well as, inhibiting the oxidation of linoleic acid.

Lastly, a multi-solvent extraction of ginger was compared to BHA and BHT

(both at 200ppm) during a six month storage of sunflower oil. Here the peroxide and free fatty acid values were documented and it was found that the ginger extract

was more effective than BHA at 1600 and 2400ppm and comparable to BHT at

2400ppm (Salariya and Habib, 2003).

2.1.2. Comparative Antioxidant Studies of Active Compounds Found in Ginger

Several conclusions have been made regarding the structure-activity relationship amongst the gingerol and shogaol analogues. Dugasani et al. (2010) showed that in the scavenging of DPPH, superoxide and hydroxyl radicals, the pattern of effectiveness is as follows: [6]-shogaol > [10]-gingerol > [8]-gingerol> [6]- gingerol, which implies that increasing the carbon chain length will increase the efficacy. However, studies done by Kikuzaki and Nakatani (1993), Masuda et al.

(2004) and Cho, K. et al. (2001) challenge this correlation.

Kikuzaki and Nakatani (1993) studied the structure activity relationships of

12 isolated antioxidants from a dichloromethane extract and compared them to that of -tocopherol. They used the FTC and TBARS methods with linoleic acid as the substrate and concluded that the increase in antioxidant activity was due to the constituents along the carbon chain and the substitution patterns on the benzene ring. In both classes of the gingerol-related structures and diarylheptanoids (Figure

3), the diacetate moieties were the strongest antioxidants. In comparing the stereoisomers having the configuration of 3R,5S versus the 3S,5S configuration, the

3R,5S moiety was also stronger. Overall, however, all 12 isolates were more effective than -tocopherol.

O OH O O

O OH HO OH O O HO O O

O OH O HO

HO OH OH O O OH OH O HO OH

HO OAc OAc

OAc OAc OR1 OR1 O HO OH

HO R2 a: R1= CH3, R2= H (3R,5S) b: R1= CH3, R2= OCH3 (3R, 5S) O O c: R1=R2=H (3S,5S) O O O O O HO Gingerol-related HO OH Diarylheptanoids

Figure 3. Isolated compounds from ginger

Masuda et al. (2004) reported testing the isolated gingerol-related compounds and diarylheptanoids for their DPPH radical scavenging activity and the inhibitory effect on the oxidation of methyl linoleate under aeration and heating using the Oil Stability Index (OSI) method. In this study, no significant differences in activity amongst the compounds of different alkyl chain lengths, in neither DPPH

nor OSI methods, were observed. However, there were differences between the efficacy of the dehydrogingerdiones and the gingerols in that, dehydrogingerdiones were weaker antioxidants in scavenging DPPH but more effective in retarding the auto-oxidation of the oil. These results again, support the inferences that the substrates and substituents on the alkyl chain contribute to the antioxidant efficacy more so than the alkyl chain length.

Lastly to contradict the correlation of increasing antioxidant strength with chain length, Cho et al. (2001) isolated [4]-gingerol, [6]-gingerol, [8]-gingerol, [10]- gingerol and [6]-shogaol from a methanol and subsequent ethyl acetate extraction.

The antioxidant strength of each isolate was tested against BHT and BHA in scavenging DPPH. It was reported that [4]-gingerol and [6]-gingerol were superior to BHT but [4]-gingerol, [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol were inferior to BHA.

Zancan et al. (2002) studied different extraction techniques along with CO2 and solvent combined CO2 extractions. They compared the chemical compositions of each, containing varying quantities of gingerols and shogaols, and determined that the extractions with higher amounts of gingerols and shogaols had a higher antioxidant response during the coupled oxidation of linoleic acid and -carotene.

They also confirmed that the gingerol and shogaol-related moieties were responsible for the increased efficacy and indicated that using co-solvents, such as, ethanol or isopropanol is advisable because it assisted the CO2 in extracting greater amounts of gingerols and shogaols.

2.2. Antioxidants in Food Applications

Oxidative degradation reactions inherent in food not only shorten shelf-life but also make food products unacceptable for the human palette. Pre-cooked, ground meats are prone to lipid oxidation especially those with a high fat content of

20-30%. To extend shelf-life, it has become a common practice to incorporate antioxidants into meat products (Biswas et al., 2001) and in response to the demand for all-natural label claims, natural extracts with known antioxidant activity have been studied. In a six-month study by Sasse et al. (2009), grape seed extract, rosemary oleoresin and a water-soluble oregano extract were compared to a few commonly used synthetic antioxidants. They were tested in pre-cooked and subsequently frozen pork patties, and the effectiveness of the antioxidants on the oxidative stability measured by TBARS, was as follows: propyl gallate > grape seed>

BHA> BHT> rosemary oleoresin> oregano water soluble extract. This indicates that natural extracts may be used in place of synthetic antioxidants in several food applications.

2.2.1. The Use of Ginger as an Antioxidant in Food

An ethanolic ginger extract made from ground ginger rhizomes was mixed with raw, ground pork meat (0.5% w/w) with which, patties were formed. The patties were roasted and kept frozen at 4°C for 21 days before the fat was extracted.

Compared to the patties without ginger extract, those with, had lower evidence of triacylglycerol hydrolysis, hydroperoxide formation and overall, lower peroxide values (Takacsova et al., 2000).

Another application involving the antioxidant effect of ginger powder has been achieved in cookie dough. Both ginger and cumin powder were tested for their antioxidant ability in scavenging DPPH using the methanolic extracts of the finely ground cookies containing one to five percent powder based on 100g of flour. The results showed a linear increase in the antioxidant activity with the increased percentage of both powders, and ginger, having a greater scavenging effect than cumin (Abdel-Samie et al., 2010). The linear increase in efficiency is also congruent with the study of ginger extract in sunflower oil and the thermal stability and antioxidant strength after heat treatment of the ginger extract (Salariya and Habib,

2003).

2.3. Citrus Instability

Citrus flavors are quite popular amongst the general population as a flavor preference; however, the main constituents, in namely lemon oil, limonene and citral, can be rather unstable. This instability results in the loss of quality and consumer acceptability and therefore, shelf-life is limited.

2.3.1. Limonene Oxidation

Limonene (4-isopropenyl-1-methyl-cyclohexene) is a monocyclic terpene and the major constituent of citrus essential oils making up over 95% of lemon peel oil (Djordjevic et al., 2007). It is a chiral molecule in which the R-enantiomer exists in nature as D-limonene ((+)-limonene) (Figure 4) and can be obtained by the steam distillation of citrus fruits.

Figure 4. D-limonene

Although limonene is relatively stable and can be distilled without decomposition, at elevated temperatures around 300°C, it will racemize forming isoprene which easily oxidizes in moist air (Karlberg et al., 1992). The flavor of limonene is fresh, green, citrusy and slightly piney, while the oxidation product, limonene 1,2-epoxide, is greener, with weedy, minty and herbaceous notes.

In the presence of acid, a hydrogen shift may occur forming a carbocation; once this happens, limonene is racemized and the chirality of all degradation products is lost. The carbocations that are formed can lead to addition products, such as, -terpineol, -terpineol and -terpineol, while the dehydration reactions of limonene can produce p-cymene (Thomas and Bessiere, 1989).

McGraw et al. (1999) studied the thermal degradation of limonene in the presence of oxygen and found that the process can be divided into several types of oxidation pathways. The first is the dehydrogenation of a six-membered ring containing one or two double bonds and in the case of limonene, thymol is produced

(Figure 5a). The second is the formation of epoxides which can produce limonene oxide (Figure 5b) and the last to mention involves the reaction of singlet oxygen which will lead to the oxidation of the carbon adjacent to the carbon-carbon double bond, known as allylic oxidation (McGraw et al., 1999).

Since 1914, it has been known that limonene is very sensitive to oxygen and it was observed that in a drum of limonene under the Australian sun, perillyl alcohol

(Figure 5c) was formed. As with the oxidation of unsaturated fatty acids, limonene oxidation initially results in the formation of hydroperoxides which can then undergo scission reactions leading to the formation of numerous products including perillyl acetate, carveol, carveol acetate and carvone (Figure 5d) (Djordjevic et al.,

2007). Degradation products of monoterpenes and their derivatives can also be traced back to the rearrangement of the skeletal structure; for example, one rearrangement product of limonene is eucarvone (Figure 5e) (McGraw et al., 1999).

OH a e

eucarvone thymol

b d O O limonene

OH carvone limonene oxide

perillyl alcohol

Figure 5. Reaction products of limonene

Syntheses using limonene as a starting reagent have been vastly studied. As early as, 1922, polymers were formed by reacting limonene with phenols and the

addition of hydrogen sulfide with limonene forms menth-1-ene-8-thiol, which is commonly known in the flavor industry as, grapefruit mercaptan (Thomas and

Bessiere, 1989).

2.3.2. Citral Degradation

Citral (3,7-dimethyl-2,6-octadienal) is an unsaturated monoterpene aldehyde with an additional -unsaturated double bond and is comprised of two geometric isomers, neral and geranial, in the ratio of 2:3 (Liang et al., 2004). Citral is the characterizing constituent in lemon oil lending a fresh and zesty aroma; however, it is known to degrade rapidly under low pH and oxidative stress conditions, yielding undesirable off-flavors.

The acid-catalyzed cyclization of citral begins with the isomerization of geranial to neral, which can form monoterpene alcohols, such as, p-menthandien-8- ol (Scheme 1). After further oxidation, p-cymene-8-ol can be formed, while dehydration reactions will produce aromatic compounds, such as, ,p-dimethylstyrene, p-cymene and p-cresol (Djordjevic et al., 2007; Ueno et al., 2006). p-Cresol and p-methylacetophenone are known to be the most offensive off-flavors of citral degradation (Djordjevic et al., 2007).

H+ + H+ O -H O citral 2 OH OH

p-menthadien-8-ols p-mentha-1,4(8),5-triene

auto-oxidation + +

OOH O p-cresol p-methyl-acetophenone 8-hydroperoxy-p-cymene

Scheme 1. Proposed mechanism for the formation of p-cresol from citral (Ueno et al., 2006)

2.3.3. Stability Efforts

Several attempts have been made to inhibit limonene from oxidizing and citral from degrading. These efforts include: reducing storage temperature, adjusting pH, modifying headspace, storage in dark containers and using antioxidants within the system itself.

Karlberg et al. (1994) added BHT to an open container (exposed to air) of limonene at room temperature and found that when the BHT was consumed, the auto-oxidation took place similarly to the samples without having any antioxidant added. However, when the sample was kept in a closed container in the dark at 4°C, the concentration of limonene remained stable for the duration of the 12 month study.

In another effort to stabilize limonene, oleoresins, such as, rosemary

(Rosmarinus officinalis L.), anise (Pinpinella anisum L.), caraway (Carum carvi L.) and dill (Anethum graveolens L.) were compared to the commonly used antioxidants: mixed tocopherols (50%), BHA and BHA/BHT (1:1) by measuring the peroxide values and monitoring the degradation markers (limonene epoxides, carveols and carvone) on the GC. Out of the oleoresins, rosemary was the most effective in inhibiting limonene oxidation; it was more effective than BHA, comparable to the mixed tocopherols and slightly less effective than the combined BHA/BHT (Lee and

Widmer, 1994).

Kimura et al. (1983) used common antioxidants, such as, BHT, BHA, n-propyl gallate, -tocopherol, nordihydroguaiaretic acid and n-tritriacontan-16,18-dione, in a citric acid solution of citral but found that none of these antioxidants were able to inhibit citral from degrading. Peacock and Kuneman (1985), however, found that the use of isoascorbic acid inhibited the formation of p-cymene-8-ol and its dehydration product, ,p-dimethylstyrene. Liang et al. (2004) found that natural antioxidants (grape seed, pomegranate seed, green tea and black tea extracts) inhibited the formation of p-methylacetophenone by blocking the pathway of p- cymene-8-ol and lastly, is the combined use of natural antioxidants in emulsion systems. In the study by Yang et al. (2011), the emulsion systems employing either

-carotene or tanshinone, were effective in diminishing the rate of citral degradation.

3. HYPOTHESIS

The antioxidants found in ginger will enhance the flavor and stability of lemon oil.

4. RESEARCH OBJECTIVES

4.1 Ginger Selection

To investigate the nine ginger powders, extraction methods, extraction solvents and antioxidant activity to determine which ginger powder should be selected for further, in-depth work.

4.2 Fractionation and ORAC Monitoring

Once the ginger powder is selected, the appropriate extraction protocol must be followed for its use in the SepBox®. Each fraction collected from the SepBox® will be tested for its ORAC value to determine which fractions are responsible for the antioxidant efficacy of the ginger extract.

4.3 Identification of Strongest Antioxidants

Select ginger fractions will be identified based on their ORAC values and the quantity available. This will be done using the developed LC/MS/UV method,

MS/MS, HRMS, as well as, referencing the literature (Jiang et al., 2007).

4.4 Use of Antioxidants in Lemon Oil

Select concentrations of each antioxidant will be employed into a single-fold

Argentinian lemon oil which will be placed in the thermal acceleration storage chamber for four weeks. Upon removal, the lemon oils will be analyzed by GC, measured for their peroxide values and tasted by an expert citrus panel in a high- acid tasting solution. The oils with antioxidants will be compared to two controls

without antioxidants: one refrigerated and one thermally accelerated sample, to determine how effective the antioxidants are at preventing the lemon oil from degrading.

5. EXPERIMENTAL

5.1. Materials

5.1.1. Ginger Powders

Synthite Industries Limited (Kerala, India) provided the following: Synthite

Rajakumari, Synthite Irutti, Synthite Nigerian and Synthite Shimoga. Whole Herb

Company (California, USA) provided large quantities of both Whole Herb Nigerian and Whole Herb India ginger powders and Buderim Ginger America, Inc (New Jersey,

USA) provided both Buderim Australian and Buderim Fijian ginger powders.

PharmaChem Chinese ginger was purchased from PharmaChem Laboratories (New

Jersey, USA.)

5.1.2. Solvents, Reagents and Supplies

Acetone (Fisher A929-4, Acetone Optima®); hexane (Fisher H302-4 HPLC

Grade); methanol (Fisher A452-4 HPLC Grade); water (Fisher W5-4 HPLC Grade); dichloromethane (Fisher D150-4 HPLC Grade). Acetone/acetonitrile/methanol

(1:1:1 by volume) and formic acid were purchased from EMD Chemicals, Inc.

(Pennsylvania, USA). The ethanol (HPLC Grade) was purchased from Pharmco-

Aaper (Connecticut/ Kentucky, USA). DPPH and hexahydrocurcumin were purchased from Sigma-Aldrich (Missouri, USA). Mixed tocopherols (50% in sunflower oil) was purchased from VitaBlend Nederland B.V. (Netherlands). [6]- gingerol, [8]-gingerol, [10]-gingerol, [6]-shogaol, [8]-shogaol and [10]-shogaol were purchased from ChromaDex (California, USA). Tetrahydrocurcumin (TetraPure®) was from Sabinsa Corporation (New Jersey, USA). [4]-Gingerol, [4]-gingerdiol, [4]- shogaol, [6]-gingerdiol and octahydrocurcumin were synthesized by IFF R&D (New

Jersey, USA). OxiSelect Oxygen Radical Antioxidant Capacity (ORAC) Activity Assay was purchased from Cell BioLabs, Inc. (California, USA). C4-Silica from Macherey-

Nagel GnmH & Co. (Berlin, Germany). Single-fold Argentinian-type lemon oil was purchased from Capua (Calabria, Italy). For the peroxide assay, the chloroform

(CHCl3, C606SK, HPLC Grade), glacial acetic acid (A35), potassium iodide (KI, P412) and 0.1 N solution of sodium thiosulfate (12427-001, Acros) were purchased from

Fisher Scientific (Pennsylvania, USA); the starch indicator (8050-16) was from Ricca

Chemical Company (Texas, USA). For the high-acid tasting solution, sodium benzoate and citric acid were purchased from Mitsubishi International (New Jersey,

USA) and the high-fructose corn syrup was purchased from Paulaur Corporation

(New Jersey, USA).

5.2. Instruments and Equipment

Mettler Toledo Model: HG63 Halogen was used to measure moisture content.

Orbital Shaker ISF-1-V; Adolf Kuhner AG, Schwez was used for the acetone extractions of ginger powders. Buchi Speed Extractor E-914 (Switzerland) was used for the hexane extractions of ginger powders. Rotary Evaporator by Buchi: Rotavapor R-

210 and Vacuum Controller V-850 (Switzerland) were used for solvent removal.

Agilent 6890 Gas Chromatograph equipped with a 5973 Mass Selective Detector was used for GC profiling of acetone extracts and for peak identification of markers in lemon oils; Agilent 7890A was used for the area percent of the identified markers in the lemon oils. The Agilent 1200 was used for HPLC profiling of ginger extracts and the DPPH assay with a Luna C18 column by Phenomenex. For structure

identification, the LC/MS/UV Thermo LSQ Mass was used and the High Resolution

Mass Spectrometer (HRMS) Thermo LTQ Orbitrap was used to obtain exact mass.

The percolator was made for IFF by ChemGlass. Beckman Coulter Spectrometer

DTX880 was used to measure the fluorescence during the ORAC assay employing a

480nm excitation filter and a 520nm emission filter. SepBox® 2D-5000 by sepiatec

(Berlin, Germany) utilizing MAC Process traps and columns (including Carbo, C4, C8 and C18.) Branson 3150 Sonicator; Thermo Scientific Centrifuge CL10; IsoTemp

Vacuum Oven Model 285A (Fisher Scientific); Biotage SP1 utilizing column KP-Sil

40+M; Spectroline Model CC-80 Ultraviolet Fluorescence Analysis Cabinet

(Spectronics Corporation) with a short wave UV of 254 nm and a long wave UV of

365 nm. Thermally Accelerated Storage Chamber “Hot Box” (Scientific Climate

Systems, Inc.)

5.3. Methods and Protocols

5.3.1. Moisture Content

All moisture contents were taken in triplicate and averaged.

5.3.2. Extraction of Ginger Powders and Fractionation of Synthite Nigerian Acetone Extract

5.3.2.1. Acetone Extracts Using Orbital Shaker

Each ginger powder (30 g) was loaded into an orbital shaker flask with acetone (270 g) and covered with aluminum foil. The samples were agitated at 150 revolutions per minute (rpm) for 72 hours at 21°C. The samples were then

decanted, filtered and the solvent was removed under mild heat (40°C) and reduced pressure (200 mmHg).

5.3.2.2. Hexane Extracts Using Speed Extractor

Using the Speed Extractor, solvents with increasing polarity, in the order of hexanes, methanol then water, were run through the ginger powder, under a set pressure (140 bar for hexane and methanol, and 40 bar for water) and a temperature of 60°C, 75°C and 96°C, respectively. The samples were decanted, filtered and the solvent was removed under mild heat (40°C) and reduced pressure

(200 mmHg).

5.3.2.3. Percolator Extract for SepBox®

600 g of Synthite Nigerian ginger powder was loaded into the percolator and

2800 mL of hexane was added. The mixture was circulated with a pump for three hours at 40°C (the temperature of the heating oil in the jacket was 52°C). The solvent was drained and concentrated to a residue, using the rotary evaporator under reduced pressure (temperature: 40°C; pressure: 200 mmHg) and labeled

“Synthite Nigerian Hexane Extract.” Yield: 22.9 g. To the spent ginger powder, 2800 mL of acetone was added into the percolator and circulated with a pump for another three hours at 40°C (the temperature of the heating oil in the jacket was 52°C). The solvent was drained and the procedure was repeated using another 2800 mL of acetone. The two extracts were combined and concentrated to a residue, using a rotary evaporator under reduced pressure (temperature: 40°C; pressure: 200

mmHg) and labeled “Synthite Nigerian Acetone Extract.” Yield: 24.2 g and the HPLC can be found in the appendix (Appendix 23b). Lastly, 2800 mL of ethanol was added to the percolator to the twice spent ginger powder and circulated with a pump at

60°C for five hours. The solvent was drained and concentrated to a residue, using a rotary evaporator under reduced pressure (temperature: 40°C; pressure: 200 mmHg) and labeled “Synthite Nigerian Ethanol Extract.” Yield: 15 g.

5.3.2.4. SepBox® Fractionation

5.033 g of Synthite Nigerian acetone extract was added to 40 mL of methanol with the assistance of sonication for 30 minutes. The dilution was centrifuged for five minutes at 3000 rpm, decanted and 20 mL of acetone was added to the residue.

The acetone dilution was centrifuged, combined with the methanol solution and filtered using a 1 m syringe filter followed by a 0.22 m syringe filter. After, the ginger methanol/ acetone solution was placed in a round bottom flask and 29.68 g of C4-silica was added; the system was sonicated and put onto the rotary evaporator to remove the solvent at a mild temperature (40°C) and reduced pressure (200 mmHg).

Approximately 30 g of ginger sample on silica was added to the injection column containing a 7 g layer of C4-silica and topped off with another 7 g of C4- silica. The injection column was flushed with water to remove any water soluble sugars, starches, etc., present in sample which directly exited the system and went into the fraction collector. The sample remaining on the column then eluted to the

main separation column in which a series of trapping and separation began; following a detailed IFF developed protocol for fractionating natural extracts.

5.3.3. Analytical Work on Ginger Extracts and Lemon Oils

5.3.3.1. Gas Chromatogram (GC) Work on Ginger Acetone Extracts

From the orbital shaker acetone extracts, 10% dilutions in acetone filtered and injected into the GC. The instrument method called for a 1 l injection, a split ratio of 50:1, with a 250°C inlet temperature, an OV1 column (50 m x 0.23 mm x 0.5

m), a carrier flow (He) of 1.0 ml/min and an oven temperature program of 40°C ramped at 2°C per minute to 310°C with a 40 minute hold.

5.3.3.2. Gas Chromatogram/Mass Spectrometry (GC/MS) Work on Lemon Oils

The GC method called for a 1 l injection of the neat oil, a split ratio of 100:1, with a 70°C inlet temperature, an OV1 column (60 m x 0.25 mm x 1 m), a carrier flow (He) of 2.0 ml/min and an oven temperature program of 70°C ramped at 3°C per minute to 220°C with a 15 minute hold.

The MS parameters were as follows: 1 l injection of neat lemon oil, a split ratio of 30:1, 70°C inlet temperature, an OV1 column (30 m x 0.25 mm x 0.25 m), a carrier flow (He) of 1.5 ml/min and an oven temperature program of 70°C ramped at 5°C per minute to 250°C with a 10 minute hold.

5.3.3.3. High Performance Liquid Chromatography (HPLC) Profiling

The HPLC profiling of each acetone and hexane extract was done using 10% dilutions in acetone. The neat hexane extracts were placed into the vacuum oven at

40°C and 0.3 mmHg overnight before diluting with acetone.

LC Conditions: Column: Luna C 18 (250 x 4.6 mm), 5 m Eluent: A= 0.1% formic acid in H20; B = 0.1% formic acid in ACN Gradient: Time %A %B Flow (mL/min) 0.00 90.0 10.0 0.7 30.00 20.0 80.0 0.7 35.00 0.0 100.0 07 36.00 90.0 10.0 0.7 40.00 90.0 10.0 0.7 Injection Volume: 10 l UV : 280 nm Solvent for the samples: Acetone

5.3.3.4. Structure Identification of Major Components in Select Ginger Fractions

A developed LC/UV/ MS method was used for the analysis of the samples and based on the HRMS, literature and MSn patterns, several compounds were tentatively proposed. All the samples were diluted with or dissolved in acetone to a concentration of 0.5%. The solutions were filtered prior to LC/MS analysis.

MS conditions: Ion Mode: APCI positive Capillary Voltage: 14 V Capillary Temperature: 225 C Discharge Current: 5 A Sheath Gas Flow: 65 arb Isolation Width: 1.5 amu Normalized Collision Energy: 35%

5.4. Antioxidant Assays

5.4.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Activity

0.05% dilutions of ginger extracts in methanol were made for each extract.

0.5 mL of the 0.05% ginger extract in methanol was added to 0.5 mL of a 0.1% dilution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) in methanol. These were compared to the blank (0.5 mL of the 0.1% DPPH dilution in methanol with 0.5 mL methanol) to serve as a reference. Three hours later, the absorbance was measured at 517 nm using the LC and the antioxidant activity were measured using the calculation below.

Antioxidant Activity (%) = (1-abs of sample/ abs of DPPH ref)*100

Surveyor LC Conditions: Eluent: A= 0.1% formic acid in H20; B = 0.1% formic acid in ACN Gradient: Time %A %B Flow (mL/min) 0.00 10 90 0.7 1.00 10 90 0.7 Temperature: ambient Detection: UV @ 517 nm Injection Volume: 10 l Solvent for the samples: Methanol

5.4.2. Oxygen Radical Absorbance Capacity (ORAC)

All 18 ginger extracts (hexane extracts from speed extractor and acetone extracts from orbital shaker) and the 316 fractions from the hexane washed acetone extract of Synthite Nigerian, were tested for their ORAC values at 0.01%; following the protocol provided by Cell Bio Labs.

5.4.3. Peroxide Value Assay

Following the protocol developed by IFF, approximately 5 g of oil is weighed into a 250 mL Erlenmeyer flask. 30 mL of an acetic acid-chloroform solution (3:2 v/v) is added, followed by the addition of 0.5 mL of saturated potassium-iodide solution and agitation for one minute. With vigorous agitation, 30 mL of distilled water is added followed by 0.5 mL of starch indicator. The flask is subsequently titrated with a 0.1 N sodium thiosulfate solution. The end-point is reached when the blue color disappears and a yellow color remains for 30 seconds. The peroxide values can be calculated using the equation below.

Peroxide Value = (S – B) (N) (1000) Weight of Oil S: Titration Volume of the Sample B: Titration Volume of the Blank N: Normality of the Sodium Thiosulfate solution

5.5. Syntheses of Ginger-related Compounds

5.5.1. [4]-Gingerol

Dissolve 4.0 g of zingerone in 100 mL of dry tetrahydrofuran (THF) and cool the mixture with a dry ice bath to -78°C. Under nitrogen (N2), add 8.5 mL of 2.5 N butyl lithium solution (dropwise) to the reaction mixture. Stir for 30 minutes at -78

°C after the addition, then add 10.0 mL of 2.0 N lithium diisopropylamide (LDA) solution (dropwise) to the mixture. Stir the mixture at -78 °C for 3h, then add a solution of 1.5 g butyraldehyde in 20 mL of dry THF (dropwise). Stir the mixture at -

78°C for 3h, remove the dry ice bath and stir at 0°C for 1h. Quench the reaction by slowly adding 100 mL of 1N hydrogen chloride (HCl). Extract the mixture with 200 mL of ether, then wash the organic layer with brine twice, and dry with magnesium sulfate (MgSO4).

After filtration and concentration, the crude was purified by flash chromatography (silica gel, ethyl acetate (EtOAc)/Hexanes). 2.5 g of product was obtained and the yield was 51%. 400-MHz 1H NMR (CDCl3) : 6.82 ppm (d, 1H, J =

7.92 Hz), 6.62-6.71 ppm (m, 2H), 5.51-5.55 (br, 1H), 4.00-4.10 (m, 1H), 3.87 ppm (s,

3H), 2.45-2.95 (m, 7H), 1.30-1.52 ppm (m, 4H), 0.92 ppm (t, 3H, J = 7.02 Hz).

5.5.2. [4]-Shogaol

Dissolve 3 g of 4-gingerol in 150 ml of THF, add 150 ml of 10% HCl solution to the flask and reflux for 4 h. Cool down and extract with 200 ml of ether, wash the organic layer with brine twice, then dry with MgSO4.

The crude was purified by flash chromatography (silica gel, EtOAc/Hexanes) in which 1.8 g of product is obtained (Yield: 64.4%). 400-MHz 1H NMR (CDCl3) :

6.65-6.85 ppm (m, 4H), 6.09 ppm (d, 1H, J = 15.89 Hz, of t, J = 1.50 Hz), 5.61 ppm (s,

1H), 3.86 ppm (s, 3H), 2.80-2.90 ppm (m, 4H), 2.17 ppm (t, 2H, J = 7.11 Hz, of d, J =

1.50 Hz), 1.43-1.53 ppm (m, 2H), 0.93 ppm (t, 3H, J = 7.45 Hz).

5.5.3. [4]-Gingerdiol

Dissolve 0.4 g of sodium borohydride (NaBH4) in 20 ml of ethanol, then cool down with an ice bath. Add the solution of 2.5 g [4]-gingerol in 10 mL of ethanol

(dropwise) to the mixture and keep the temperature around 0°C. After addition, remove cooling bath and stir at room temperature for 2 h. Cool down again to 0°C, add 10 mL of 1 N HCl (dropwise) to decompose any excess of NaBH4. Transfer the

mixture to a separatory funnel, add 100 ml of brine, and extract with 200 ml of

EtOAc. Wash the organic layer with brine twice and dry with MgSO4.

The crude was purified by flash chromatography (silica gel, EtOAc/Hexanes) in which, 2.0 g of product is obtained (Yield: 79%). 400-MHz 1H NMR (CDCl3) : 6.82 ppm (d, 1H, J = 7.92 Hz), 6.62-6.71 ppm (m, 2H), 5.54 ppm (s, 1H), 3.87 ppm (s, 3H),

3.60-4.40 (br, m, 2H), 2.50-3.20 (m, 3H), 1.30-1.90 ppm (m, 9H), 0.85-0.95 ppm (m,

3H).

5.5.4 [6]-Gingerdiol

O OH OH OH O NaBH4 O EtOH HO HO

Dissolve 0.32 g of NaBH4 in 20 ml of ethanol, cool down with an ice bath. Add the solution of 2.5 g of [6]-gingerol in 10 mL of ethanol (dropwise) to the mixture and keep the temperature around 0°C. After addition, remove the cooling bath and stir at room temperature for 4 h. Cool down again to 0°C, add 10 mL of 1 N HCl

(dropwise) to decompose any excess of NaBH4. Transfer the mixture to a separatory funnel, add 100 ml of brine, and extract with 200 ml of EtOAc. Wash the organic layer with brine twice and dry with MgSO4.

The crude was purified by flash chromatography (silica gel, EtOAc/Hexanes),

1.2 g of product is obtained and the yield is 47.7%. 400-MHz 1H NMR (CDCl3) :

6.77-6.84 ppm (m, 1H), 6.62-6.74 ppm (m, 2H), 5.56 ppm (br. s, 1H), 3.83-4.34 ppm

(m, 2H), 3.87 ppm (4s, 3H), 2.57-2.79 ppm (m, 2H), 1.37-1.92 ppm (m, 6H), 1.15-

1.37 ppm (m, 6H), 0.83-0.91 (m, 3H). 400-MHz 1H NMR (CDCl3) of the starting material, [6]-gingerol: 6.81 ppm (d, 1H, J = 8.00 Hz), 6.67 ppm (s, 1H, J = 1.92 Hz),

6.64 ppm (d, 1H, J = 8.04 Hz, of d, J = 1.92 Hz), 5.73 ppm (br. s, 1H), 3.86 ppm (br. s,

1H), 3.85 ppm (3s, 3H), 3.04 ppm (br. s, 1H), 2.80-2.86 ppm (m, 2H), 2.70-2.76 ppm

(m, 2H), 2.45-2.59 ppm (m, 2H), 1.23-1.50 ppm (m, 8H), 0.88 (t, 3H, J = 6.88 Hz).

5.5.5. Octahydrocurcumin

O O O O HO OH HO OH NiCl2, NaBH4

EtOH O O OH OH

Dissolve 5 g of curcumin in 250 ml of ethanol and 56 mL of water. Start stirring, add 3.23 g of nickel chloride (NiCl2) to the mixture and cool down to 0 °C.

Under N2 atmosphere, add 4.11 g of sodium borohydride to the mixture within 1 h.

After stirring at 0 °C for 5 h, neutralize the reaction mixture with 0.4 N HCl to pH 4.

Transfer the mixture to a separatory funnel and extract with 200 ml of dichloromethane. Wash the organic layer with brine twice and dry with MgSO4.

After filtration and concentration, 6.3 g of crude product is obtained.

After purifying 1 g using the BioTage (an automated preparative HPLC), 0.41 g was purified to 80% purity. 400-MHz 1H NMR (CD3OD) : 6.77 ppm (d, 2H, J = 1.72

Hz), 6.73 ppm (d, 2H, J = 8.00 Hz), 6.64 ppm (d, 2H, J = 8.04 Hz, of d, J = 1.76 Hz),

3.83 ppm (m, 2H), 3.80 ppm (s, 6H), 2.54-2.73 ppm (m, 4H), 1.69-1.78 ppm (m, 4H),

1.56-1.60 ppm (m, 2H).

5.6. Sensory Evaluation of Lemon Oils

5.6.1. Sensory Evaluation of Lemon Oils after Thermal Acceleration

After being stored in the thermally accelerated storage chamber at 90.7°F for four weeks, each lemon oil was tasted at 100ppm in a high-acid tasting solution with a pH of 2.56. The panel consisted of five experts in the field: Dennis Kujawski

[Director of Flavor Creations], Richard Dandrea [Senior Flavorist], Hedy Kulka

[Senior Flavorist], Anusha Sampath [Flavorist Trainee] and Sharon Tortola [Junior

Flavorist]. Each flavorist was given a list of sensory attributes (specifically pertaining to citrus) and was asked to rate each attribute on the scale from “0” to

“9”, with “0” representing no flavor impact perceived and “9” representing the greatest flavor impact imaginable.

5.6.2. Sensory Evaluation of Lemon Beverages after Thermal Acceleration

Each lemon oil (of set one) was added to a high-acid tasting solution with a pH of 2.56 at 100 ppm and tasted after being stored in the thermally accelerated storage chamber at 90.7°F for two weeks. The panel consisted of seven experts in the field: Dennis Kujawski [Director of Flavor Creations], Richard Dandrea [Senior

Flavorist], Hedy Kulka [Senior Flavorist], Anusha Sampath [Flavorist Trainee],

Sharon Tortola [Junior Flavorist] and myself, Kathryn Bardsley [Junior Flavorist].

Each flavorist was given a list of sensory attributes (specifically pertaining to citrus) and was asked to rate each attribute on the scale from “0” to “9”, with “0” representing no flavor impact perceived and “9” representing the greatest flavor impact imaginable.

5.7. Statistical Analysis of Lemon Oils and Peroxide Values

5.7.1. Statistical Analysis of Peroxide Values

The data analyses were performed using the One-Way ANOVA using JMP's Fit

Model, with the peroxide value being the dependent and the samples of oils as the independent variables. The Post-Hoc was the Student's T-Tests with a significance of a 95% Confident Level (p=0.05). The same was used for the paired comparisons of the lemon oils: set one versus set two.

5.7.2. Statistical Analysis of Tasting Results

The data analyses were performed using the Two-Way ANOVA using JMP's Fit

Model, with the ratings being the dependent and the samples of oils and panelists as the independent variables (using JMP's default effects). The Post-Hoc was the

Student's T-Tests with a significance of a 95% Confident Level (p=0.05).

6. RESULTS AND DISCUSSION

6.1. General Background Information

6.1.1. Growing Regions of Ginger Samples

All (nine) ginger powders are Zingiber officinale Roscoe and are discussed below with their growing regions and harvesting methods.

“Synthite Rajakumari” is grown in the state of Kerala, India, at an altitude around 3000 to 5000 feet. Once the ginger is harvested, it is dried on rocks and the skin is peeled manually. “Synthite Irutti” is obtained from low altitude areas of

Kerala (north side) and is sold mostly with the skin on. “Synthite Shimoga” is obtained from a high altitude area in the neighboring state Karnataka in north east

India. All of the before mentioned rhizomes were harvested between December of

2009 and February of 2010; after which, the ginger is washed and dried.

“Synthite Nigerian” is grown in Kafanja, northern Nigeria, in the Kaduna area and was harvested in February of 2010. The ginger grown here is typically sold by

Nigeria for extraction, grinding and/ or trade and is purchased in its dried, whole form after it has been cleaned for stones and filth and subsequently ground. The ground ginger does not contain carriers or additives and is processed in Synthite’s factory.

“Whole Herb Nigerian” ginger is collected from various regions by local farmers from the following five states of the Federation namely, Kaduna, Nasarawa,

Benue, Niger and Gombe, with Kaduna being the major producer. The ginger was harvested between January and April in 2009. “Whole Herb Indian” ginger is grown

in Kerala and was also harvested between January and April in 2010. The rhizomes are air dried, peeled and ground to a fine powder without the use of carriers.

“Buderim Australian” ginger is grown in Queensland which is about 100-130 miles from Brisbane and was harvested in 2008. “Buderim Fijian” ginger is grown within 40 miles from the plant, located in Suva, Fiji and was harvested in 2009. The ginger is harvested around late June/ early July, dried using drum-drying and subsequently ground, without the use of carriers.

Lastly, “PharmaChem Chinese” ginger is grown in China, however, no additional information was provided.

6.1.2. Moisture Content of Ginger Powders

Whole Whole Pharma-

Sample Synthite Synthite Synthite Synthite Herb Herb Buderim Buderim Chem

Rajakumari Irutti Nigerian Shimoga Nigerian Indian Australian Fijian Chinese

Average

Moisture 11.63% 11.22% 10.14% 10.58% 8.88% 10.89% 8.97% 9.42% 11.45%

Content

Table 1. Average moisture content of ginger powders

The moisture content of Whole Herb Nigerian was the lowest value out of the set, followed by the two Buderim samples, Australian and Fijian. Of the nine ginger powders, the three with the lowest moisture contents are also the three that were harvested the earliest (between 2008 and 2009); therefore, dehydration is likely occurring during storage. In addition, from the processing information received from the vendors, Buderim was the only company that reported the use drum- drying, which may also contribute to the low moisture content values.

6.1.3. Sensory Descriptions of Ginger Samples

6.1.3.1. Aroma of Dried Ginger Powders

Synthite Rajakumari: dark brown notes, cooked ginger, spicy, Indian food, fresh, warm.

Synthite Irutti: chocolate, sweet, ginger snaps, herbal, turmeric-like, cocoa, slightly fruity, citrus, cheap chocolate.

Synthite Nigerian: sharp citrus, fresh, pungency of fresh ginger, lemon-lime, candied, fruity, soft profile.

Synthite Shimoga: earthy, mushroom, moldy, cheesy, pungent with citrus undertones, muddy, less fresh, dirty, raw mushrooms, unwashed lettuce, fishy, ammonia.

Whole Herb Nigerian: spicy, savory, sage, chicken rub, citrus, very unique, powdery, lacks freshness, common ginger, slight vitamin note.

Whole Herb Indian: freeze dried, refrigerator smell, slight candied note, cooked ginger, lacks freshness, herbal, slightly dirty, slight mushroom.

Buderim Australian: woody, sandalwood, aroma reminiscent of an old church, balanced, not fresh, sugary, Nestea iced tea mix, choking.

Buderim Fijian: typical ginger powder, hay, not fresh, cosmetic, lemon juice, soft, lacks pungency.

PharmaChem Chinese: fresh, clean, slight citrus, lemon-lime, ginger, hay, slightly animalic, pungent, earthy, turmeric-like, cinnamon, nasal warming, cooked ginger, methional, chocolate undertones, butyraldehyde, valeraldehyde.

6.1.3.2. Blotter Aroma of 10% Dilutions in Acetone

Synthite Rajakumari: sulfurous, citrus, marigold, asafetida, liver, brussels sprout, alliaceous, green, vegetable, processed meat.

Synthite Irutti: household cleaner, limonene, perfume, nutty, walnut hulls, earthy, cocoa bean, more volatile than the others, drying, malt, heavy, fixative.

Synthite Nigerian: herbaceous, furfural, sweet, gingerbread cookies, maltol, citronellal, waxy at end, warm, cinnamic, baker’s cinnamon, resinous, pungency of mustard powder (thiocyanate-like), eugenol, anise.

Synthite Shimoga: leafy, wet leaves, green fatty aldehydes, slight mint, coumarin, cardboard, pencil shavings, cedrol, grease/ fat, slight citrus.

Whole Herb Nigerian: animalic, less fresh smelling than Synthite’s Nigerian ginger, phenolic, cured meat, not reminiscent of ginger, spice house, earthy, old dried herbs, woody, cardamom, masculine, citral, very lemony.

Whole Herb Indian: ginger powder, slight terpeney, black pepper-like, maple, sweet potato, methional, brown, candied, powdery.

Buderim Australian: fermented, animalic (like a gutted deer and digested grass), musky, hay, grainy, dried fruit (Craisins), slight civet, cat urine.

Buderim Fijian: sweaty, brown, caramel, musty, hay, broom, furfural, coumarin, fatty acids, slight cinnamon notes, slight guaiacol on dry down.

PharmaChem Chinese: geraniol, citrus, fresh ginger, phenolic (Band-aid), pungent, slight eugenol, typical ginger character, end of profile is similar to beginning of

Synthite Nigerian with warm, spicy notes.

6.1.3.3. Tasting Comments of 0.1% Solutions in Water

Synthite Rajakumari: lime, heat builds significantly, fecal, manure, skatol, animalic, processed meats.

Synthite Irutti: capsicum heat, woody.

Synthite Nigerian: warm, brown ginger, warming, tingle.

Synthite Shimoga: citral, floral, a lot of heat, stemmy, woody.

Whole Herb Nigerian: floral, much less heat than Synthite’s Nigerian, bland, delayed tingle.

Whole Herb Indian: perfume, soapy, weak, slightly warming.

Buderim Australian: very floral, old ginger (not fresh tasting), moderate heat, coumarinic, hay.

Buderim Fijian: brown, perfume, sand, a lot of heat, bitter, tingle.

PharmaChem Chinese: typical ginger flavor, fast onset of heat/ tingle on tip of tongue, flat.

After compiling the aroma, flavor and taste descriptors of each, one may conclude that discernable differences amongst sensory attributes may suggest differences in the chemical composition between the ginger samples. In addition, one may be able to taste which ginger has the highest concentration of gingerols and shogaols based on a greater sensation of heat (ie. Synthite Nigerian) and lastly, is the favorable pairing of ginger with citrus, as a few (ie. Whole Herb Nigerian and

Synthite Shimoga), have inherent lemon-like characteristics.

6.1.4. Extraction of Ginger Powders

6.1.4.1. Acetone Extracts Using Orbital Shaker

Orbital Whole Whole Pharma-

Shaker Synthite Synthite Synthite Synthite Herb Herb Buderim Buderim Chem

Acetone Rajakumari Irutti Nigerian Shimoga Nigerian Indian Australian Fijian Chinese

Extract

Yield;

Percent 1.51g; 1.43g; 1.81g; 1.64g; 2.04g; 1.59g; 1.31g; 1.5g; 1.61g;

Yield 5.03% 4.77% 6.03% 5.47% 6.8% 5.3% 4.37% 5% 5.37%

Table 2. Yields of acetone extracts using orbital shaker

6.1.4.2. Hexane Extracts Using Speed Extractor

Speed

Extractor Synthite Synthite Synthite Synthite Whole Whole Buderim Buderim Pharma-

Hexane Rajakumari Irutti Nigerian Shimoga Herb Herb Australian Fijian Chem

Extract Nigerian Indian Chinese

Starting

Material 30.68g 35.07g 28.46g 28.39g 30.21g 25.76g 44g 32.31g 30.74g

Yield;

Percent 1.09g; 1.02g; 1.08g; 1.62g; 1.19g; 1.03g; 0.84g; 0.94g; 1.12g;

Yield 3.55% 3.58% 3.79% 5.71% 3.94% 4% 1.91% 2.91% 3.64%

Table 3. Yields of hexane extracts using speed extractor

6.1.5. Analytical Work: GC and HPLC

The profiling of each ginger sample has been completed by both gas chromatography (GC) and high-performance liquid chromatography (HPLC). The

GC results for the acetone extracts are listed alphabetically and by retention time, along with their chromatograms, and can be found in the appendix (Appendices 1-

11). In addition, the HPLC chromatograms of both hexane and acetone extracts can also be found in the appendix (Appendices 12-20 and 21-29, respectively). Both profiles of the GCs and HPLCs are comparable to one another and the profiles of the ginger samples themselves are similar, except for the two Buderim ginger powders.

Both Buderim samples were deficient in -zingiberene, which is probably a result of the processing conditions concerning drum-drying.

6.2. Ginger Selection

6.2.1. Determination of Gingerol and Shogaol Content

To compare the gingerol and shogaol content of each, the peak areas (from the HPLC chromatograms) of [6]-, [8]- and [10]-gingerols and [6]-, [8]- and [10]- shogaols were added together. To simplify the data, the summations are ranked in the table below from “1” to “9”, with “1” representing the largest summation of gingerols and shogaols and “9” representing the smallest (Table 4). (Please see

Appendix 30 for the peak areas of each.)

It was determined that Buderim Fijian ginger, followed by Synthite Nigerian, had the highest amount of gingerols and shogaols out of the hexane extracts. For the acetone extracts, both Nigerian samples (Synthite and Whole Herb Company, respectively) had the largest summation.

Ranking of Whole Whole Pharma-

Gingerols & Synthite Synthite Synthite Synthite Herb Herb Buderim Buderim Chem

Shogaols Rajakumari Irutti Nigerian Shimoga Nigerian Indian Australian Fijian Chinese

Hexane

Peak Areas 8 9 2 4 3 6 7 1 5

Acetone

Peak Areas 6 5 1 8 2 7 9 4 3

Table 4. Ranking of gingerol and shogaol summation

6.2.2. Antioxidant Activity Studies

6.2.2.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Activity

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay is a method used to determine the efficiency of antioxidants in scavenging free radicals. DPPH is a stable free radical, violet in color and has an absorbance of 517nm. Below depicts the scheme for the assay and demonstrates two methods of termination: the hydrogen donating ability of an antioxidant (AH) and the association of two radicals, which results in the reduction of the DPPH radical. Once the DPPH radical is reduced, the absorbance will decrease and turn yellow in color (Brand-Williams et al., 1995).

DPPH• + AH  DPPH-H + A•

DPPH• + A•  DPPH-A

Scheme 2. The reduction of DPPH

From the results listed in Table 5, the acetone extracts were able to scavenge

DPPH to a greater extent than the hexane extracts. The acetone extract of Synthite

Nigerian was able to scavenge nearly 88% of the DPPH radical while the hexane extract was able to scavenge 55.4%. The highest scavenging activity for the hexane extracts was 60% by Whole Herb Nigerian.

DPPH Whole Whole Pharma-

Scavenging Synthite Synthite Synthite Synthite Herb Herb Buderim Buderim Chem

Activity (%) Rajakumari Irutti Nigerian Shimoga Nigerian Indian Australian Fijian Chinese

Hexane

Extracts 21.9 25.4 55.4 45.8 60 50.3 40.1 55.4 41.5

Acetone

Extracts 57.8 55.7 87.9 39 32.9 33 56.4 50.9 59

Table 5. DPPH scavenging activity of ginger extracts

6.2.2.2. Oxygen Radical Absorbance Capacity (ORAC)

The ORAC assay is based on fluorescence which is quenched by peroxyl radicals. To determine the effectiveness of an antioxidant, the inhibition time to prolong the quenching of the fluorescent probe is measured.

The concentrations of the ginger extracts were selected by employing a range of levels from 0.0001% to 5% to determine the best fit of the decay curve. The

ORAC fluorescence curves of each ginger extract at 0.01% can be found in the appendix (Appendices 31 and 32) while Table 6 depicts the values of antioxidant activity.

Whole Whole Pharma-

ORAC Synthite Synthite Synthite Synthite Herb Herb Buderim Buderim Chem

Values Rajakumari Irutti Nigerian Shimoga Nigerian Indian Australian Fijian Chinese

Hexane

Extracts 2.20 0.44 1.19 1.90 2.46 1.94 0.10 2.38 1.73

Acetone

Extracts 2.89 3.55 5.39 2.34 4.50 1.31 0.79 3.87 3.72 Table 6. ORAC values for hexane and acetone extracts at 0.01%

6.2.2.3. Summary of Hexane Extracts versus Acetone Extracts

Hexane Peak Area Peak Area DPPH DPPH ORAC ORAC

Extracts Values Ranking Values Ranking Values Ranking Synthite

Rajakumari 38399 8 21.9 9 2.20 3 Synthite

Irutti 34409 9 25.4 8 0.44 8 Synthite

Nigerian 64578 2 55.4 2/3 1.19 7 Synthite

Shimoga 57718 4 45.8 5 1.90 5 Whole Herb

Nigerian 61472 3 60 1 2.46 1 Whole Herb

Indian 52244 6 50.3 4 1.94 4 Buderim

Australian 42023 7 40.1 7 0.10 9 Buderim

Fijian 68996 1 55.4 2/3 2.38 2 Pharma-

Chem 56474 5 41.5 6 1.73 6

Chinese Table 7. Compilation of hexane extracts: values and rankings

Acetone Peak Area Peak Area DPPH DPPH ORAC ORAC

Extracts Values Ranking Values Ranking Values Ranking Synthite

Rajakumari 83808 6 57.8 3 2.89 6 Synthite

Irutti 88144 5 55.7 5 3.55 5 Synthite

Nigerian 99649 1 87.9 1 5.39 1 Synthite

Shimoga 79563 8 39 7 2.34 7 Whole Herb

Nigerian 93751 2 32.9 9 4.50 2 Whole Herb

Indian 80246 7 33 8 1.31 8 Buderim

Australian 69144 9 56.4 4 0.79 9 Buderim

Fijian 91112 4 50.9 6 3.87 3 Pharma-

Chem 93515 3 59 2 3.72 4

Chinese Table 8. Compilation of acetone extracts: values and rankings

Tables 7 and 8 depict all before-mentioned values, as well as, the assigned rankings for discussion. In Table 7, the ranking of hexane peak areas parallel with the ranking of DPPH but are less aligned with the ORAC results. In Table 8, the ranking of acetone peak areas parallel with the ranking of ORAC but are less aligned with DPPH. One can see how effective gingerols and shogaols are in both assays, as the acetone extracts have a greater concentration and exude greater DPPH scavenging, along with higher ORAC values. However, the discrepancies between the ORAC and DPPH rankings for each solvent, demonstrates that other

constituents, with differing polarities, are being extracted along with the gingerols and shogaols which may enhance the antioxidant efficacy of ginger.

After observing the peak area summations along with the results of the antioxidant assays, the acetone extract of Synthite Nigerian ginger was selected to be studied more in depth.

6.3. Fractionation and ORAC Monitoring

6.3.1 . Ginger Extract Fractionation via SepBox®

The SepBox® system is a unique approach in fractionating plant material. It is a two dimensional HPLC which combines preparative-scale HPLC with solid phase extraction (SPE). The SepBox® is fully automated and can fractionate up to five grams of plant material within 24 hours covering the entire spectrum of polarities and recovering sufficient material for structure elucidation. The method employed was developed by IFF for the fractionation of botanical extracts, which requires a reversed-phase column; therefore, the selected ginger extract had to be first washed with hexanes before extracting with acetone. The hexane washed acetone extract of

Synthite Nigerian yielded 316 fractions with high purity.

6.3.2. ORAC Assay of Each Fraction

Each of the 316 fractions was tested for its ORAC value and the top 10 fractions having the highest ORAC values were listed in Table 9.

Top 10 Fraction Number ORAC Value 1 126 11.55 2 111 11.48 3 313 11.48 4 142 11.4 5 167 11.39 6 310 11.36 7 308 11.33 8 127 11.32 9 312 11.29 10 164 11.25 Table 9. Synthite Nigerian SepBox® fractions with the highest ORAC values

Also included, are the ORAC values of known antioxidants found in ginger

(Table 10). The concentrations reported refer to the concentrations added to the assay; however, after all other reagents are added to each well, the concentration of the extract is diluted to 12.5% of the initial concentration.

Table 10 demonstrates how antioxidant activity is strongly dependent on concentration; all activities of solutions greater than 0.1% and below 0.01% diminish greatly while the range between 0.01 and 0.1% proves to be optimum. The diminished activity of the 1% dilutions also supports the theory that when a concentration is too high, an antioxidant will become a “pro-oxidant.” Last to mention is the diminished activity of the antioxidants when increasing their chain length which directly supports the contradiction made earlier: increasing the chain length does not necessarily increase the antioxidant activity.

Initial Final

Concentration Concentration [6]- [6]- [10]- [10]-

Added to Well in Well Gingerol Shogaol Gingerol Shogaol

1% 0.125% 8.85 5.14 5.94 -0.72

0.1% 0.0125% 11.08 11.18 8.39 8.30

0.01% 0.00125% 11.16 11.21 6.40 5.81

0.001% 0.000125% 3.61 1.11 -1.09 -1.39

Table 10. ORAC values for known ginger antioxidants

6.3.3. Identification of Antioxidants

6.3.3.1 Proposed Active Compounds

Select ginger fractions were chosen for identification based on their ORAC values and the quantity available. Using the developed LC/MS/UV method, MS/MS,

HRMS and referencing the literature (Jiang et al., 2007), several compounds were proposed as the major components of the chosen fractions. The following fractions were among the top 10 but could not be used for identification due to sample size:

F313, 310, 308 and 312. The most abundant fraction was F166 which was used to discern the selected concentration of 5000 ppm for analysis and was later found to be [6]-gingerol. Fractions 126, 111, 142, 167, 110, 165 and 108 were identified

(Figures 6a-i; Table 11) and the corresponding LC and exact mass chromatograms can be found in the appendix (Appendices 33-35).

a. F126/ F111: Hexahydrocurcumin (5-hydroxy-1,7-bis(4-hydroxy-3- methoxyphenyl)- 3-heptanone)

OMe OMe

HO OH OH O

CH 2 CH 2 CH CH 2 C CH 2 CH 2

b. F142: [4]-Gingerdiol ((3R,5S)- 1-(4-hydroxy-3-methoxyphenyl)-3,5- octanediol)

OH OH

MeO R S Pr-n

c. F167: [6]-Gingerol ((5S)- 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3- decanone)

O OH

Me S (CH 2 ) 4

OMe

d. F110-1: 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3- methoxyphenyl)-3,5-heptanediol

OMe OMe

HO OH OH OH

CH 2 CH 2 CH CH 2 CH CH 2 CH 2 MeO

e. F110-2: Octahydrocurcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5 heptanediol)

OH OH

CH 2 CH 2 CH CH 2 CH CH 2 CH 2

HO OH

OMe OMe

f. F165-1: [6]-Gingerdiol ((3S,5R)- 1-(4-hydroxy-3-methoxyphenyl)-3,5- decanediol)

OH OH

Me S R (CH 2 ) 4

OMe

g. F165-2: [6]-Gingerol ((5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3- decanone)

O OH

Me S (CH 2 ) 4

OMe

h. F108-1: 1-(3,4-dihydroxy-5-methoxyphenyl)-5-hydroxy-7-(4- hydroxy-3-methoxyphenyl)-3-heptanone)

OMe OMe

HO OH O OH

CH 2 CH 2 C CH 2 CH CH 2 CH 2 HO

i. F108-2: 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-(4-hydroxy-3- methoxyphenyl)-3-heptanone

O OH

CH 2 CH 2 C CH 2 CH CH 2 CH 2

HO OH

OMe OH

Figure 6. Structures (a-i) of the identified compounds

Fraction Retention Molecular Number Time (min) (M+H)+ APCI (ms/ms)+ Formula Identification 126 18.87 405.1911 207 C22H28O7 F126-1* 19.56 375.1806 177 C21H26O6 Hexahydrocurcumin 111 18.87 405.1911 207 C22H28O7 F111-1* = F126-1* 142 16.91 297.206 279, 261 C17H28O4 F142-1* 17.98 341.2324 323, 305, 287 C19H32O5 F142-2* 19.2 269.1748 251, 233, 177, 163 C15H24O4 [4]-Gingerdiol 167 25.72 491.2285 431, 371, 177 C26H34O9 F167-1* 26.14 295.1902 277, 177 C17H26O4 [6]-Gingerol 110 18.33 407.2058 371, 389, 193, 167 C22H30O7 F110-1* 19.17 377.1955 341, 359, 163, 217 C21H28O6 Octahydrocurcumin 165 26.04 297.2058 279, 261, 163, 137 C17H28O4 [6]-Gingerdiol 27.33 295.1904 277, 177 C17H26O4 [6]-Gingerol 108 18.01 391.1749 373, 193 C21H26O7 F108-1* 18.26 361.1645 343, 179 C20H24O6 F108-2* *Names were too long to include in table but can be found next to their corresponding structures.

Table 11. Active compounds identified from Synthite Nigerian ginger fractions

6.3.3.2 Confirmed Active Compounds

The racemic structures of the proposed compounds: hexahydrocurcumin,

[6]-gingerol, [4]-gingerdiol, octahydrocurcumin and [6]-gingerdiol from ginger fractions F126/ 111, F167, F142, F110 and F165, respectively, were confirmed using commercial standards (hexahydrocurcumin and [6]-gingerol) and an IFF synthetic standards ([4]-gingerdiol, octahydrocurcumin and [6]-gingerdiol).

For F126/ 111 and F167, based on LC/MS/MS, HRMS and information reported in literature, were proposed to be hexahydrocurcumin and [6]-gingerol,

respectively. In terms of LC retention time and MS2 spectra, the peak of F126/111 and that of F167, matched very well with the commercial standards and the proposed structures were confirmed. The LC/MS/MS profiles and 1H-NMR spectra can be found in the appendix (Appendices 36- 39).

The same has been done for F142, F110 and F165; based on LC/MS/MS,

HRMS and information obtained from literature, the synthetic isomers of the standards matched very well (Appendices 40-45).

6.4. Use of Antioxidants in Lemon Oil

6.4.1 Determination of Antioxidant Concentrations via ORAC

Based on the ORAC values for the known antioxidants of ginger (Table 10), a range of concentrations to be added to the wells were selected for each antioxidant:

[4]-gingerol, [4]-shogaol and [4]-gingerdiol (0.001% to 1%), [6]-gingerol and [6]- gingerdiol (0.006% to 1%), S-[6]-gingerol (0.008 to 0.6%), tetrahydrocurcumin, hexahydrocurcumin and octahydrocurcumin (0.0001 to 1%) and mixed tocopherols

(0.00006 to 0.1%). (Please refer to Appendix 46 for the levels employed in the assay with their corresponding ORAC values.) Although tetrahydrocurcumin, [4]-gingerol and [4]-shogaol were not identified as being one of the top 10 antioxidants, because they are analogs of those identified, they too have been included in the study (1H-

NMR Appendices 47-49).

6.4.2 Antioxidants in Lemon Oil

A single-fold Argentinian lemon oil, cold-pressed by Brown extraction, was chosen for the stability study. The following table (Table 12) depicts the selected levels of antioxidants added to the lemon oil. The sets were created in triplicate and stored in amber glass bottles at 90.7°F. Two control sets were also created: one placed in the thermally accelerated storage chamber with the other samples and the other, kept in the refrigerator. All samples were removed after four weeks.

Concentration in Lemon Oil Concentration in Lemon Oil Antioxidant: (Set 1): (Set 2): 4-gingerol 12.5 ppm 100 ppm 4-gingerdiol 12.5 ppm 100 ppm 4-shogaol 12.5 ppm 100 ppm 6-gingerol 125 ppm 1000 ppm S-6-gingerol 125 ppm 6-gingerdiol 75 ppm 600 ppm 6-shogaol 125 ppm 8-gingerol 125 ppm 8-shogaol 125 ppm 10-gingerol 125 ppm 10-shogaol 125 ppm Tetrahydrocurcumin 50 ppm 400 ppm Hexahydrocurcumin 125 ppm Octahydrocurcumin 25 ppm 200 ppm Mixed Tocopherols 250 ppm 1000 ppm Table 12. Levels of antioxidants added to Argentinian lemon oil

6.4.3 Peroxide Study of the Lemon Oils with and without Ginger-related Antioxidants

The starting oil has an average peroxide value of 12.6 meq (milliequivalents of peroxides per 1000 g of oil), in which, a peroxide value up to 20 meq is deemed acceptable by taste. Table 13 depicts the mean peroxide value for each sample.

Set 1: Set 2: Concentration Peroxide Concentration Peroxide of Antioxidant Value of of Antioxidant Value of Antioxidant: in Lemon Oil: Lemon Oil: in Lemon Oil: Lemon Oil: Blank Control, Refrigerated 15.86 meq Blank Control, Thermally Accelerated 19.1 meq 4-gingerol 12.5 ppm 21.84 meq 100 ppm 20.2 meq 4-gingerdiol 12.5 ppm 22.87 meq 100 ppm 24.59 meq 4-shogaol 12.5 ppm 21.72 meq 100 ppm 22.66 meq 6-gingerol 125 ppm 23.04 meq 1000 ppm 38.78 meq S-6-gingerol 125 ppm 34.79 meq 6-gingerdiol 75 ppm 21.4 meq 600 ppm 35.48 meq 6-shogaol 125 ppm 25.65 meq 8-gingerol 125 ppm 25.32 meq 8-shogaol 125 ppm 21.03 meq 10-gingerol 125 ppm 24.18 meq 10-shogaol 125 ppm 26.69 meq Tetrahydrocurcumin 50 ppm 18.68 meq 400 ppm 30.8 meq Hexahydrocurcumin 125 ppm 21.49 meq Octahydrocurcumin 25 ppm 29.49 meq 200 ppm 28.53 meq Mixed Tocopherols 250 ppm 15.26 meq 1000 ppm 25.96 meq Table 13. Mean peroxide values of Argentinian lemon oils

For the first set, the samples containing mixed tocopherols and tetrahydrocurcumin were the most effective at prohibiting peroxide formation.

However, the increasing peroxide values for the samples containing antioxidants versus the thermally accelerated blank control, indicates that the concentration of antioxidants employed are too high.

For the second set, all levels employed were too high; however, compared to the other samples in this set, 4-gingerol, 4-shogaol and 4-gingerdiol were most

effective. When optimizing the concentrations in the ORAC assay (Appendix 46), 4- gingerol, 4-shogaol and 4-gingerdiol did not vary greatly which is evident in the lemon oil study as well; employing nearly ten times the antioxidant yielded nearly the same peroxide value (concentrations in set one versus set two).

In contrast yet supporting the statement made earlier, the results of tocopherols (250 ppm versus 1000 ppm) proves that using a concentration of an antioxidant well above optimum efficacy results in the generation of a pro-oxidant.

Another interesting observation is the linear relationship between peroxide value and ethanol content. The samples S-6-gingerol (from set 1), 6-gingerol, 6- gingerdiol and tetrahydrocurcumin (from set 2) have elevated levels of ethanol and significantly higher peroxide values (Appendices 50 and 51). This indicates that there may be trace metals in the ethanol which may have a catalytic effect triggering auto-oxidation or ethanol itself, may be a pro-oxidant.

Through statistical data analysis, it was determined that there weren’t significant differences between the control oils (refrigerated blank and thermally accelerated blank) after heat treatment, however, there were significant differences between the thermally accelerated control oil and a few samples containing antioxidants. Tables 14 through 16 were included to display the degree of significance between all peroxide values of the oils; the bar charts corresponding to these tables can be found in the appendix (Appendices 52-54). Please note, that samples sharing a common letter in the column labeled “Multiple Comparisons” denotes that there is no significant difference between them.

N Multiple Antioxidant Rows Mean Std Errors Comparisons Tocopherols 3 15.26 2.65 i Blank, Refrigerated 15.86 Tetrahydrocurcumin 3 18.68 0.98 hi Blank, Hot Box 2 19.10 2.58 ghi 8-shogaol 3 21.03 0.71 fgh 6-gingerdiol 3 21.40 1.77 efgh Hexahydrocurcumin 3 21.49 1.37 efgh 4-shogaol 3 21.72 0.49 defgh 4-gingerol 3 21.84 1.31 defgh 4-gingerdiol 3 22.87 0.56 cdefg 6-gingerol 3 23.04 0.93 cdefg 10-gingerol 3 24.18 0.72 cdef 8-gingerol 3 25.32 1.96 cde 6-shogaol 3 25.65 1.38 bcd 10-shogaol 3 26.69 1.25 bc Octahydrocurcumin 3 29.49 1.31 b S-6-gingerol 3 34.79 1.68 a p < 0.05: significant difference between the two samples at p=0.05 (95% confidence); NS : No significant difference at p>0.05 Table 14. Data analysis of peroxide values from first set of lemon oils

Multiple Antioxidant N Rows Mean Std Errors Comparisons 4-gingerol 3 20.20 1.62 f 4-shogaol 3 22.66 0.28 ef 4-gingerdiol 3 24.59 1.24 def Tocopherols 3 25.96 1.00 cde Octahydrocurcumin 2 28.53 0.11 cd Tetrahydrocurcumin 3 30.80 1.40 bc 6-gingerdiol 3 35.48 2.14 ab 6-gingerol 3 38.78 3.30 a p < 0.05: significant difference between the two samples at p=0.05 (95% confidence); NS : No significant difference at p>0.05 Table 15. Data analysis of peroxide values from second set of lemon oils

N Paired Antioxidant Rows Mean Std Errors Comparisons Blank, Hot Box 2 19.10 2.58 Blank, Refrigerated 15.86 4-gingerdiol (12.5ppm) 3 22.87 0.56 NS (p=0.28) 4-gingerdiol (100ppm) 3 24.59 1.24 4-gingerol (12.5ppm) 3 21.84 1.31 NS (p=0.47) 4-gingerol (100ppm) 3 20.20 1.62 4-shogaol (12.5ppm) 3 21.72 0.49 NS (p=0.17) 4-shogaol (100ppm) 3 22.66 0.28 6-gingerdiol (75ppm) 3 21.40 1.77 p=0.01 6-gingerdiol (0.06%) 3 35.48 2.14 6-gingerol (125ppm) 3 23.04 0.93 p=0.01 6-gingerol (0.1%) 3 38.78 3.30 Octahydrocurcumin (25ppm) 3 29.49 1.31 NS (p=0.61) Octahydrocurcumin (200ppm) 2 28.53 0.11 Tetrahydrocurcumin (50ppm) 3 18.68 0.98 p=0.00 Tetrahydrocurcumin (400ppm) 3 30.80 1.40 Tocopherols (250ppm) 3 15.26 2.65 p=0.02 Tocopherols (0.1%) 3 25.96 1.00 p < 0.05: significant difference between the two samples at p=0.05 (95% confidence); NS : No significant difference at p>0.05 Table 16. Paired comparison of significance between sets of lemon oils

As stated earlier, 6-gingerdiol, 6-gingerol and tetrahydrocurcumin from the second set, have elevated amounts of ethanol and from the statistical analysis, one can see that this increase has a significantly negative impact.

Regarding 4-gingerdiol, 4-gingerol and 4-shogaol, there is no significant difference which proves that the concentration level for these is less sensitive.

Appendix 46 depicts that changing the concentration from 40 ppm to 10,000 ppm does not yield a significant change in ORAC value. Similarly to the “four moieties”, the peroxide values of octahydrocurcumin demonstrate no significant difference between 25 ppm and 200 ppm which parallels the ORAC values (Appendix 46) at

200 ppm and 1600 ppm (representing 12.5 % of the concentration employed into

the oil). This is not the case, however, regarding tocopherols as one can see in Table

16; increasing the level beyond its optimum concentration drastically increases the peroxide value.

6.4.4. Sensory Validation of Ginger-related Antioxidants in Lemon Drink

The following attributes of the lemon oils (Table 17) were rated by an expert citrus panel consisting of five to seven flavorists, on a scale from “0” to “9”, with “0” representing no flavor impact perceived and “9” representing the greatest flavor impact imaginable.

Descriptors: Peely/ Lemon Peel Citral Candied Juicy Oxidized Cooked Plastic/ Phenolic Cherry-like/ Medicinal Piney Soapy/ Green Turpentine Table 17. Lemon beverage descriptors

The lemon oils with a peroxide value closest to the mean peroxide value of the set were selected for tasting. All oils were dosed into the high-acid tasting medium at 100 ppm.

6.4.4.1. Sensory Validation of Lemon Oils after Thermal Treatment

From the data analysis of the sensory ratings, it was determined that there were no significant differences overall, between the refrigerated lemon oil and the

oils stored in the chamber after four weeks, therefore, the complete list of tasting results has not been reported. However, there were significant differences between the oils regarding the single attribute of citral (Figure 7). Interestingly, the most favored tasting solutions were those incorporating tocopherols and tetrahydrocurcumin which were also the two oils with the lowest peroxide values.

Figure 7. Citral ratings of lemon oils after storage

6.4.4.2. Sensory Validation of Lemon Beverages after Thermal Treatment

A second tasting was conducted by a trained panel consisting of seven flavorists, using the same oils at 100ppm but after the high-acid tasting solutions containing the oils were stored in the chamber for an additional two weeks.

Representing the three control beverages, the following terms were used: hot box, refrigerated, and fresh, which refer to the lemon beverage stored in the thermally accelerated chamber for two weeks, the lemon beverage stored in the refrigerator for two weeks and the lemon beverage made on the morning of the tasting, respectively; none of which, contained antioxidants.

For the attribute, peely, the fresh and refrigerated samples were significantly more peely than the others but not significantly different from each other. In addition, none of the samples containing antioxidants were significantly more peely than the hot box sample (Table 18).

Attribute Sample Mean Std Err Significance Peely Fresh Blank 6.43 0.48 a Peely Refrigerated Blank 6.00 0.76 a Peely 4-Gingerol 12.75ppm 4.71 0.47 b Peely 6-Shogaol 123.5ppm 4.29 0.78 bc Peely Hexahydrocurcumin 125ppm 4.29 0.64 bc Peely 4-Gingerdiol 12.5ppm 4.14 0.55 bc Peely 4-Shogaol 13.5ppm 3.86 0.83 bcd Peely 10-Shogaol 125ppm 3.71 0.99 bcd Peely 6-Gingerdiol 79.7ppm 3.71 0.78 bcd Peely Mixed Tocopherols 497ppm 3.71 0.61 bcd Peely Octahydrocurcumin 26.7ppm 3.71 0.71 bcd Peely Hot Box Blank 3.57 0.61 bcd Peely Tetrahydrocurcumin 51.25ppm 3.43 0.75 cd Peely 6-Gingerol 126ppm 3.29 0.68 cd Peely 8-Shogaol 123.7ppm 3.29 0.52 cd Peely 10-Gingerol 122.4ppm 3.14 0.77 cd Peely 8-Gingerol 121ppm 2.71 0.64 d Table 18. Statistical analysis of lemon beverage tastings for the attribute, peely

For citral, only hexahydrocurcumin and octahydrocurcumin had a significantly greater citral impact than the hot box sample. All other samples containing antioxidants had significantly less citral flavor than the refrigerated sample and were not significantly different from the hot box sample. Lastly, the refrigerated and fresh samples were significantly different from one another which depicts how unstable citral is in an acidic environment (Table 19).

Std Attribute Sample Mean Err Significance Citral Fresh Blank 6.86 0.34 a Citral Refrigerated Blank 5.43 0.69 b Citral Hexahydrocurcumin 125ppm 4.29 0.52 c Citral Octahydrocurcumin 26.7ppm 4.00 0.58 cd Citral 10-Shogaol 125ppm 3.86 0.74 cde Citral 4-Shogaol 13.5ppm 3.86 0.86 cde Citral 8-Shogaol 123.7ppm 3.71 0.61 cdef Citral 4-Gingerol 12.75ppm 3.57 0.48 cdef Citral 6-Gingerdiol 79.7ppm 3.57 0.87 cdef Citral 6-Shogaol 123.5ppm 3.57 0.57 cdef Citral Tetrahydrocurcumin 51.25ppm 3.43 0.53 cdef Citral 10-Gingerol 122.4ppm 3.29 0.61 cdef Citral Mixed Tocopherols 497ppm 3.29 0.64 cdef Citral 4-Gingerdiol 12.5ppm 3.14 0.67 def Citral 8-Gingerol 121ppm 2.86 0.51 ef Citral Hot Box Blank 2.86 0.59 ef Citral 6-Gingerol 126ppm 2.71 0.84 f Table 19. Statistical analysis of lemon beverage tastings for the attribute, citral

For the attribute, candied, 4-gingerol, 6-gingerdiol and 8-shogaol were as candied as the refrigerated sample and significantly different from the hot box. The remaining samples were not significantly different from the hot box sample but 6- shogaol, 10-shogaol, 4-shogaol, tocopherols, 10-gingerol, 6-gingerol and hexahydrocurcumin were not significantly different from the refrigerated sample either. All samples, including the refrigerated sample, were significantly less candied compared to the fresh lemon beverage (Table 20).

Std Attribute Sample Mean Err Significance Candied Fresh Blank 6.00 0.58 a Candied Refrigerated Blank 4.86 0.74 b Candied 4-Gingerol 12.75ppm 4.71 0.52 bc Candied 6-Gingerdiol 79.7ppm 4.29 0.78 bcd Candied 8-Shogaol 123.7ppm 4.29 0.64 bcd Candied 6-Shogaol 123.5ppm 4.14 0.55 bcdef Candied 10-Shogaol 125ppm 4.00 0.62 bcdef Candied 4-Shogaol 13.5ppm 4.00 0.82 bcdef Candied Mixed Tocopherols 497ppm 4.00 0.58 bcdef Candied 10-Gingerol 122.4ppm 3.86 0.59 bcdef Candied 6-Gingerol 126ppm 3.86 0.80 bcdef Candied Hexahydrocurcumin 125ppm 3.86 0.51 bcdef Candied 4-Gingerdiol 12.5ppm 3.71 0.87 cdef Candied Octahydrocurcumin 26.7ppm 3.57 0.65 def Candied Tetrahydrocurcumin 51.25ppm 3.29 0.52 def Candied Hot Box Blank 3.14 0.74 ef Candied 8-Gingerol 121ppm 3.00 0.72 f Table 20. Statistical analysis of lemon beverage tastings for the attribute, candied

As depicted in Table 21, 4-gingerol and hexahydrocurcumin were juicier than the hot box sample and not significantly different than the refrigerated sample. In addition, 4-gingerol was the only sample besides the refrigerated sample that was as juicy as the fresh. Out of the remaining samples, only 6-shogaol was as juicy as both the hot box and refrigerated samples, while all other samples were significantly different from the refrigerated sample and not significantly different from the hot box sample.

Std Attribute Sample Mean Err Significance Juicy Fresh Blank 4.71 0.52 a Juicy Refrigerated Blank 4.43 0.57 ab Juicy 4-Gingerol 12.75ppm 3.57 0.75 abc Juicy Hexahydrocurcumin 125ppm 3.43 0.72 bcd Juicy 6-Shogaol 123.5ppm 3.29 0.64 bcde Juicy 10-Shogaol 125ppm 3.14 0.40 cde Juicy 4-Gingerdiol 12.5ppm 2.86 0.74 cde Juicy 4-Shogaol 13.5ppm 2.86 0.70 cde Juicy Mixed Tocopherols 497ppm 2.86 0.77 cde Juicy 10-Gingerol 122.4ppm 2.71 0.52 cde Juicy 6-Gingerdiol 79.7ppm 2.71 0.71 cde Juicy 8-Shogaol 123.7ppm 2.71 0.47 cde Juicy Octahydrocurcumin 26.7ppm 2.71 0.81 cde Juicy Tetrahydrocurcumin 51.25ppm 2.71 0.36 cde Juicy 6-Gingerol 126ppm 2.57 0.65 cde Juicy 8-Gingerol 121ppm 2.29 0.68 de Juicy Hot Box Blank 2.14 0.40 e Table 21. Statistical analysis of lemon beverage tastings for the attribute, juicy

For the attribute, oxidized, none of the samples containing antioxidants were significantly different from the hot box sample; however, 4-gingerdiol, 10-shogaol,

6-gingerdiol, 4-shogaol, octahydrocurcumin, 6-gingerol, 6-shogaol, 4-gingerol and hexahydrocurcumin were not significantly more oxidized than the refrigerated sample. All beverages, including the refrigerated lemon beverage, were significantly more oxidized than the fresh, which depicts how sensitive a panelist are towards perceiving oxidation (Table 22).

Std Attribute Sample Mean Err Significance Oxidized 8-Gingerol 121ppm 5.14 0.74 a Oxidized 10-Gingerol 122.4ppm 4.43 0.95 ab Oxidized Tetrahydrocurcumin 51.25ppm 4.29 0.71 abc Oxidized Hot Box Blank 4.14 1.12 abc Oxidized 8-Shogaol 123.7ppm 4.00 0.62 abc Oxidized Mixed Tocopherols 497ppm 3.71 0.81 abc Oxidized 4-Gingerdiol 12.5ppm 3.57 0.78 bcd Oxidized 10-Shogaol 125ppm 3.43 0.75 bcd Oxidized 6-Gingerdiol 79.7ppm 3.29 1.02 bcd Oxidized 4-Shogaol 13.5ppm 3.14 0.70 bcd Oxidized Octahydrocurcumin 26.7ppm 3.14 0.70 bcd Oxidized 6-Gingerol 126ppm 3.00 0.93 bcd Oxidized 6-Shogaol 123.5ppm 3.00 0.65 bcd Oxidized 4-Gingerol 12.75ppm 2.86 0.86 cd Oxidized Hexahydrocurcumin 125ppm 2.86 0.86 cd Oxidized Refrigerated Blank 2.14 0.77 d Oxidized Fresh Blank 0.14 0.14 e Table 22. Statistical analysis of lemon beverage tastings for the attribute, oxidized

In Table 23, 6-shogaol, hexahydrocurcumin and 10-shogaol were significantly less cooked tasting than the hot box sample but not significantly worse than the refrigerated sample. 4-Gingerdiol was the only sample that was not significantly different from either, the hot box or the refrigerated sample.

Std Attribute Sample Mean Err Significance Cooked Hot Box Blank 4.86 0.70 a Cooked Mixed Tocopherols 497ppm 4.43 0.65 ab Cooked Octahydrocurcumin 26.7ppm 4.43 0.53 ab Cooked 10-Gingerol 122.4ppm 4.29 0.78 ab Cooked 8-Gingerol 121ppm 4.29 0.71 ab Cooked 4-Shogaol 13.5ppm 4.14 0.59 ab Cooked 6-Gingerdiol 79.7ppm 4.14 0.77 ab Cooked 6-Gingerol 126ppm 4.14 0.46 ab Cooked 8-Shogaol 123.7ppm 4.14 0.46 ab Cooked 4-Gingerol 12.75ppm 4.00 0.93 abc Cooked Tetrahydrocurcumin 51.25ppm 4.00 0.79 abc Cooked 4-Gingerdiol 12.5ppm 3.86 0.77 abcd Cooked 6-Shogaol 123.5ppm 3.57 0.78 bcd Cooked Hexahydrocurcumin 125ppm 3.00 0.62 cd Cooked Refrigerated Blank 3.00 1.02 cd Cooked 10-Shogaol 125ppm 2.86 0.67 d Cooked Fresh Blank 1.14 0.70 e Table 23. Statistical analysis of lemon beverage tastings for the attribute, cooked

For the attribute, plastic or phenolic, all samples, excluding the fresh sample, were statistically similar to the hot box sample; however, octahydrocurcumin, 10- shogaol and the refrigerated, were the only samples that were not significantly different from the fresh sample (Table 24).

Std Attribute Sample Mean Err Significance Plastic/ Phenolic 10-Gingerol 122.4ppm 2.29 1.08 a Plastic/ Phenolic 4-Shogaol 13.5ppm 2.29 0.94 a Plastic/ Phenolic 6-Shogaol 123.5ppm 2.14 0.96 a Plastic/ Phenolic 6-Gingerdiol 79.7ppm 2.00 0.87 a Plastic/ Phenolic 6-Gingerol 126ppm 2.00 0.95 a Plastic/ Phenolic Tetrahydrocurcumin 51.25ppm 2.00 0.82 a Plastic/ Phenolic 4-Gingerol 12.75ppm 1.86 0.59 a Plastic/ Phenolic 8-Gingerol 121ppm 1.86 0.86 a Plastic/ Phenolic Mixed Tocopherols 497ppm 1.86 0.80 a Plastic/ Phenolic 4-Gingerdiol 12.5ppm 1.71 0.71 a Plastic/ Phenolic 8-Shogaol 123.7ppm 1.71 0.71 a Plastic/ Phenolic Hot Box Blank 1.71 0.94 a Plastic/ Phenolic Hexahydrocurcumin 125ppm 1.57 0.81 a Plastic/ Phenolic Octahydrocurcumin 26.7ppm 1.43 0.81 ab Plastic/ Phenolic 10-Shogaol 125ppm 1.29 0.64 ab Plastic/ Phenolic Refrigerated Blank 1.14 0.86 ab Plastic/ Phenolic Fresh Blank 0.14 0.14 b Table 24. Statistical analysis of lemon beverage tastings for the attribute, plastic/phenolic

For cherry or medicinal, none of the samples were significantly different from the hot box except for the refrigerated and fresh sample. 8-gingerol, 6-gingerdiol,

10-gingerol and 6-gingerol, however, had significantly more cherry and medicinal off-notes than the refrigerated sample. In addition, hexahydrocurcumin, 6-shogaol,

10-shogaol, 4-gingerdiol and 4-shogaol were the only samples to not have significant differences compared to the fresh lemon beverage (Table 25).

Std Attribute Sample Mean Err Significance Cherry/ Medicinal 8-Gingerol 121ppm 2.00 0.76 a Cherry/ Medicinal 6-Gingerdiol 79.7ppm 2.00 0.85 a Cherry/ Medicinal 10-Gingerol 122.4ppm 2.00 0.90 a Cherry/ Medicinal Hot Box Blank 1.86 0.70 a Cherry/ Medicinal 6-Gingerol 126ppm 1.86 0.96 a Cherry/ Medicinal Octahydrocurcumin 26.7ppm 1.57 0.75 ab Cherry/ Medicinal Mixed Tocopherols 497ppm 1.57 0.65 ab Cherry/ Medicinal Tetrahydrocurcumin 51.25ppm 1.43 0.57 ab Cherry/ Medicinal 8-Shogaol 123.7ppm 1.43 0.61 ab Cherry/ Medicinal 4-Gingerol 12.75ppm 1.43 0.69 ab Cherry/ Medicinal Hexahydrocurcumin 125ppm 1.29 0.64 abc Cherry/ Medicinal 6-Shogaol 123.5ppm 1.29 0.81 abc Cherry/ Medicinal 10-Shogaol 125ppm 1.29 0.57 abc Cherry/ Medicinal 4-Gingerdiol 12.5ppm 1.14 0.59 abc Cherry/ Medicinal 4-Shogaol 13.5ppm 1.00 0.49 abc Cherry/ Medicinal Refrigerated Blank 0.57 0.43 bc Cherry/ Medicinal Fresh Blank 0.29 0.29 c Table 25. Statistical analysis of lemon beverage tastings for the attribute, cherry/medicinal

Other than octahydrocurcumin, 6-gingerol, 6-shogaol, 8-shogaol and tocopherols, all samples containing antioxidants had significantly less piney off- flavor than the hot box sample. In addition, except for 4-shogaol and 4-gingerol, the remaining samples were statistically equivalent to the fresh lemon beverage (Table

26).

Std Attribute Sample Mean Err Significance Piney Hot Box Blank 3.86 0.83 a Piney Octahydrocurcumin 26.7ppm 3.29 0.81 ab Piney 6-Gingerol 126ppm 2.86 0.59 abc Piney 6-Shogaol 123.5ppm 2.86 0.26 abc Piney 8-Shogaol 123.7ppm 2.86 0.70 abc Piney Mixed Tocopherols 497ppm 2.86 0.51 abc Piney 4-Shogaol 13.5ppm 2.71 0.57 bc Piney 4-Gingerol 12.75ppm 2.57 0.61 bc Piney 10-Gingerol 122.4ppm 2.43 0.72 bcd Piney 6-Gingerdiol 79.7ppm 2.43 0.57 bcd Piney Refrigerated Blank 2.43 0.61 bcd Piney Tetrahydrocurcumin 51.25ppm 2.43 0.37 bcd Piney 10-Shogaol 125ppm 2.29 0.71 bcd Piney Hexahydrocurcumin 125ppm 2.29 0.71 bcd Piney 4-Gingerdiol 12.5ppm 2.14 0.63 cd Piney 8-Gingerol 121ppm 2.00 0.58 cd Piney Fresh Blank 1.43 0.30 d Table 26. Statistical analysis of lemon beverage tastings for the attribute, piney

For the attribute, soapy or green, only 10-shogaol, hexahydrocurcumin and 6- shogaol were significantly different from the hot box sample, however, none (except for the hot box sample) were significantly different from neither the fresh nor the refrigerated sample (Table 27).

Std Attribute Sample Mean Err Significance Soapy/ Green Hot Box Blank 2.57 0.84 a Soapy/ Green 10-Gingerol 122.4ppm 2.00 0.85 ab Soapy/ Green 4-Gingerol 12.75ppm 2.00 0.62 ab Soapy/ Green 6-Gingerdiol 79.7ppm 2.00 0.82 ab Soapy/ Green 6-Gingerol 126ppm 2.00 0.82 ab Soapy/ Green 8-Shogaol 123.7ppm 1.86 0.86 ab Soapy/ Green Mixed Tocopherols 497ppm 1.86 0.59 ab Soapy/ Green Refrigerated Blank 1.86 0.80 ab Soapy/ Green Tetrahydrocurcumin 51.25ppm 1.86 0.59 ab Soapy/ Green 4-Shogaol 13.5ppm 1.71 0.57 ab Soapy/ Green 8-Gingerol 121ppm 1.71 0.84 ab Soapy/ Green Octahydrocurcumin 26.7ppm 1.71 0.64 ab Soapy/ Green 4-Gingerdiol 12.5ppm 1.57 0.72 ab Soapy/ Green 10-Shogaol 125ppm 1.29 0.42 b Soapy/ Green Hexahydrocurcumin 125ppm 1.29 0.36 b Soapy/ Green Fresh Blank 1.14 0.46 b Soapy/ Green 6-Shogaol 123.5ppm 1.00 0.31 b Table 27. Statistical analysis of lemon beverage tastings for the attribute, soapy/green

For the attribute, turpentine, octahydrocurcumin, tetrahydrocurcumin, 8- shogaol, 10-shogaol and hexahydrocurcumin were all significantly less turpentine- like in flavor compared to the hot box. In addition, 8-shogaol, the refrigerated sample, 10-shogaol and hexahydrocurcumin were also not significantly different from the fresh sample which demonstrates favorable stabilization of the lemon oils by these antioxidants (Table 28).

Std Attribute Sample Mean Err Significance Turpentine Hot Box Blank 3.14 0.99 a Turpentine Mixed Tocopherols 497ppm 3.14 0.86 a Turpentine 4-Gingerol 12.75ppm 3.00 0.76 ab Turpentine 6-Gingerol 126ppm 2.57 0.84 abc Turpentine 6-Gingerdiol 79.7ppm 2.43 0.65 abc Turpentine 6-Shogaol 123.5ppm 2.43 0.53 abc Turpentine 4-Gingerdiol 12.5ppm 2.29 0.75 abc Turpentine 10-Gingerol 122.4ppm 2.14 0.40 abc Turpentine 8-Gingerol 121ppm 2.14 0.59 abc Turpentine 4-Shogaol 13.5ppm 2.00 0.58 abcd Turpentine Octahydrocurcumin 26.7ppm 1.71 0.57 bcde Turpentine Tetrahydrocurcumin 51.25ppm 1.71 0.61 bcde Turpentine 8-Shogaol 123.7ppm 1.57 0.53 cdef Turpentine Refrigerated Blank 1.43 0.81 cdef Turpentine 10-Shogaol 125ppm 0.71 0.29 def Turpentine Hexahydrocurcumin 125ppm 0.57 0.20 ef Turpentine Fresh Blank 0.29 0.18 f Table 28. Statistical analysis of lemon beverage tastings for the attribute, turpentine

Overall, tocopherols and tetrahydrocurcumin had the lowest peroxide values out of the set but did not lend significant flavor stability to the lemon beverage after being stored in the thermal chamber. The most significant positive differences, however, were observed in the samples containing hexahydrocurcumin and 4- gingerol, which also parallels the peroxide study by having two of the lowest peroxide values out of the set. 10-shogaol and 6-shogaol, were second to hexahydrocurcumin and 4-gingerol, but had two of the highest peroxide values. The spider chart below (Figure 8) depicts the overall flavor perception of the six before- mentioned samples containing antioxidants compared to the three controls and clearly supports the inferences made above.

Figure 8: Flavor attributes of most preferred antioxidants in lemon beverages

6.4.5. GC Analysis of Markers Representing Limonene Oxidation and Citral Degradation

The following markers (Table 29) were used to identify which sample of lemon oil was subject to the greatest amount of degradation (Schieberle and Grosch,

1989; Djordjevic et al., 2007; Yang et al., 2011). However, the following markers were not identified in the analysis: cis-p-mentha-2,8-dien-1-hydroperoxide, trans-p- mentha-2,8-dien-1-hydroperoxide, p-mentha-1,8-dien-4-hydroperoxide, trans-p- mentha-[1(7),8]-dien-2-hydroperoxide, p-mentha-1,8-dien-3-hydroperoxide, trans- p-mentha-6,8-dien-2-hydroperoxide, cis-p-mentha-6,8-dien-2-hydroperoxide, -2- carene, eucarvone, myrtenal, trans-carveol, perillaldehyde, cis-p-mentha-[1(7),8]- dien-2-hydroperoxide. In addition, all the following had peak areas of zero: butanoic acid, 2-heptanone, 1-octen-3-ol, p-cresol, ,p-dimethylstyrene, p-

menthadien-8-ol, thymol, perillyl alcohol, p-cymeme-8-ol and p-methyl acetophenone.

Literature Degradation Products: ,p-Dimethylstyrene p-Cymene p-Methyl acetophenone p-Cresol -Terpineol 2-Heptanone 1-Octen-3-ol -2-Carene Butanoic acid 1,2-Epoxy-p-menth-8-ene Myrtenal trans-Carveol Carvone Perillyl Alcohol Perillaldehyde Limonene Oxide cis-p-Mentha-2,8-dien-1-hydroperoxide trans-p-Mentha-2,8-dien-1-hydroperoxide p-Mentha-1,8-dien-4-hydroperoxide trans-p-Mentha-[1(7),8]-dien-2-hydroperoxide p-Mentha-1,8-dien-3-hydroperoxide trans-p-Mentha-6,8-dien-2-hydroperoxide cis-p-Mentha-6,8-dien-2-hydroperoxide cis-p-Mentha-[1(7),8]-dien-2-hydroperoxide Table 29. Degradation markers for GC/MS analysis of lemon oils

The identified peaks of the tasted lemon oils can be found in Table 30 and the peak areas of all oils, including the GC of the starting oil at t=0, can be found in the appendix (Appendix 55 and 56).

p- Limonene - Lemon Oil Limonene Geranial Neral Citral Carvone Sample Cymene oxide Terpineol Control at t=0 0.19 66.34 0 0.02 2.02 1.17 3.19 0 Control Fridge D 0.28 66.52 0.01 0.02 1.96 1.13 3.09 0.03 Control Hot Box B 0.81 66.43 0.06 0.02 1.94 1.11 3.05 0.03 4-Gingerol C 0.57 66.19 0.04 0.02 1.88 1.1 2.98 0.02 4-Gingerdiol B 0.55 66.17 0.04 0.02 1.9 1.11 3.01 0.02 4-Shogaol C 0.68 66.22 0.05 0.02 1.86 1.09 2.95 0.03 6-Gingerol B 0.53 66.15 0.04 0.02 1.92 1.12 3.04 0.02 6-Gingerdiol A 0.7 66.23 0.06 0.02 1.97 1.14 3.11 0.03 6-Shogaol A 0.73 66.13 0.06 0.02 1.96 1.13 3.09 0.03 8-Gingerol B 0.73 66.19 0.06 0.02 1.89 1.1 2.99 0.02 8-Shogaol A 0.65 66.17 0.06 0.02 1.87 1.09 2.96 0.03 10-Gingerol C 0.76 66.18 0.06 0.02 1.87 1.09 2.96 0.03 10-Shogaol B 0.63 66.22 0.04 0.02 1.76 1.03 2.79 0.02 Tetrahydro- curcumin B 0.73 66.34 0.06 0.02 1.86 1.08 2.94 0.03 Hexahydro- curcumin B 0.76 66.53 0.06 0.02 1.79 1.04 2.83 0.03 Octahydro- curcumin A 0.64 65.75 0.06 0.02 1.79 1.05 2.84 0.03 Tocopherols A 0.58 66.46 0.06 0.02 1.92 1.01 2.93 0.03 Table 30. Peak areas of degradation markers of tasted lemon oils

The inconsistent expectation of the decreased amount of limonene in the control oil at t=0 compared to the heat treated samples is because limonene co- elutes with 1,8-cineole. Therefore, the level of limonene should not be used to compare the oils to one another but rather, limonene oxide instead (Table 30). The two oils with the highest citral peak area are 6-gingerdiol and 6-shogaol; however, the two samples with the highest citral ratings from the first and second tastings were tocopherols and tetrahydrocurucmin (Figure 7), and hexahydrocurcumin and octahydrocurcumin (Table 19), respectively. These findings demonstrate the complexity of the lemon oil and how several constituents may contribute to the perception of a single flavor attribute. Therefore, sensory data has a greater

significance in reflecting the flavor perception than the markers of degradation via the GC.

7. CONCLUSION

To conclude, several observations have been made. The growing region of each ginger powder affects the chemical composition, in which, Nigerian ginger was found to be superior to Australian, Chinese, Fijian and Indian ginger. In addition, processing conditions affect the chemical composition of the botanical and these differences are discernable by taste.

The extraction method also impacts the end result. In compiling the gingerol and shogaol peak areas, one can see that the hexane extracts contain less gingerols and shogaols than the acetone extracts and the acetone extracts have greater values in both assays as compared to the hexane extracts. However, if gingerols and shogaols were the only active participants, the ORAC and DPPH results would align.

Their differences indicate that there are more or less polar constituents relative to the gingerols and shogaols that are being extracted and are effective in quenching peroxyl radicals and in scavenging DPPH.

It was determined that four weeks in the thermally accelerated storage chamber was not enough time to produce significant changes in oil degradation.

However, increased antioxidant concentrations produced significant increases in peroxide levels, which prove how critical employing the correct concentration is for maximum effectiveness. In addition, it was observed that samples containing increased volumes of ethanol also had significantly higher peroxide values. To discern whether it is the ethanol itself or the presence of trace metals within the

ethanol catalyzing auto-oxidation, additional work must be completed which has been added to the ‘future work’ section of this composition.

Lastly, after tabulating the sensory results of the beverages after two weeks of storage in the thermally accelerated chamber, hexahydrocurcumin had the most positive impact in stabilizing the flavor quality, which also paralleled with its ORAC and peroxide value. In contrast, however, the mixed tocopherols had the lowest peroxide value and was one of the least preferred lemon beverages. With this said, solely relying on data from objective analyses, such as peroxide values, GC markers,

HPLC profiles, ORAC values, DPPH scavenging activity, etc. is not dependable because there are gaps behind the understanding of what and how we taste.

Therefore, sensory data is the most complete and reliable data to discern the overall impact an ingredient will have on the subjected flavor profile. To conclude, the identified antioxidants found in Synthite Nigerian ginger had a significantly positive influence on the flavor and stability of lemon oil.

8. FUTURE PERSPECTIVES

Referencing a quote from Paul Schulick, “…an isolated element, thereby exclude[s] the value of the whole herb and the synergy or inherent cooperation [the] wholeness represents” leads me to my future work.

I will repeat the lemon oil stability study and store the samples for a longer period of time, while decreasing the levels of the antioxidants. I will also add new samples to test including the acetone extract of Synthite Nigerian, BHT and ethanol

(with and without EDTA to chelate trace metals). Once effective levels are established, I will work with combinations of antioxidants to determine if there are synergistic effects and if they are more powerful that those alone.

In addition, I’d welcome the opportunity to follow the referenced work done by Yang et al. (2011) by employing, single or combined antioxidants, in an emulsion system and lastly, I plan to continue identifying those ginger fractions with the highest ORAC values.

9. REFERENCES

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Cho, K. J.; Kim, J. W.; Choi, I. L.; Kim, J. B.; Hwang, Y. S. Isolation, identification and determination of antioxidant in ginger (Zingiber officinale) rhizome. Agric. Chem. Biotechnol. 2001, 44, 12-15.

Connell, D.W. Chemistry of the essential oil and oleoresin of ginger (Zingiber officinale). Flavour Ind. 1970, 10, 677-693. (Cited from Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber, CRC Press, 2005.)

Djordjevic, D.; Cercaci, L.; Alamed, J.; McClements, D.J.; Decker, E.A. Chemical and physical stability of citral and limonene in sodium dodecyl sulfate-chitosan and gum arabic-stabilized oil-in-water emulsions. J. Agric. Food Chem. 2007, 55(9), 3585- 3591.

Dugasani, S.; Pichika, M. R.; Nadarajah, V. D.; Balijepalli, M. K.; Tandra, S.; Korlakunta, J. N. Comparative antioxidant and anti-inflammatory effects of [6]-gingerol, [8]- gingerol, [10]-gingerol and [6]-shogaol. J. Ethnopharm. 2010, 127, 515-520.

Farrell, K.T. Spices, condiments and seasonings. The AVI Pub. Co. Westport, CN, USA, 1985. (Cited from Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber, CRC Press, 2005.)

Ghasemzadeh, A.; Jaafar, H. Z. E.; Rahmat, A. Antioxidant activities, total phenolics and flavonoids content in two varieties of Malaysia young ginger (Zingiber officinale Roscoe). Molecules 2010, 15, 4324-4333.

Jayachandran, B.K.; Bai, M.M.; Salam, M.A.; Mammen, M.K.; Mathew, K.P. Performance of ginger under shade and open conditions. Indian Cocoa Arecanut Spices J., 1991, 15(2), 40-41. (Cited from Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber, CRC Press, 2005.)

Jiang, H.; Timmermann, B.N.; Gang, D.R. Characterization and identification of diarylheptanoids in ginger ( Zingiber officinale Rosc.) using high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Comm. in Mass Spectrometry. 2007, 21(4), 509-518.

Jitoe, A.; Masuda, T.; Tengah, I. G. P.; Suprapta, D. N.; Gara, I. W.; Nakatani, N. Antioxidant activity of tropical ginger extracts and analysis of the contained curcuminoids. J. Agric. Food Chem. 1992, 40, 1337-1340.

Karlberg, A-T.; Magnusson, K.; Nilsson, U. Air oxidation of D-limonene (the citrus solvent) creates potent allergens. Contact Dermatitis. 1992, 26(5), 332-340.

Karlberg, A-T.; Magnusson, K.; Nilsson, U. Influence of an anti-oxidant on the formation of allergenic compounds during auto-oxidation of D-limonene. Ann. Occup. Hyg. 1994, 38(2), 199-207.

Kikuzaki, H.; Nakatani, N. Antioxidant effects of some ginger constituents. J. Food Sci. 1993, 58, 1407-1410.

Kimura, K.; Nishimura, H.; Iwata, I.; Mizutani, J. Deterioration mechanism of lemon flavor. 2. Formation of off-odor substances arising from citral. J. Agric. Food Chem. 1983, 31(4), 801-804.

Lee, H.S.; Widmer, W.W. Evaluation of commercial oleoresins for inhibition of limonene oxidation. Proc. Fla. State Hort. Soc. 1994, 107, 281-284.

Liang, C.P.; Wang, M.; Simon, J.E.; Ho, C.T. Antioxidant activity of plant extracts on the inhibition of citral off-odor formation. Mol. Nutr. Food Res. 2004, 48, 308-317.

Mahindru, S.N. Spices in Indian Life. Sultan Chand & Sons, Delhi, 1982. (Cited from Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber, CRC Press, 2005.)

Masuda, Y.; Kikuzaki, H.; Hisamoto, M.; Nakatani, N. Antioxidant properties of gingerol related compounds from ginger. BioFactors. 2004, 21, 293-296.

McGraw, G.; Hemingway, R.; Ingram Jr., L.; Canady, C.; McGraw, W. Thermal degradation of terpenes: camphene, -carene, limonene and -terpinene. Environ. Sci. Tech. 1999, 33, 4029-4033.

Mckee, R.H.; Tometsko.A.M. Inhibition of promutagen activation by the antioxidants butylated hydroxyanisole and butylated hydroxytoluene. J. Natl. Cancer Inst. 1979, 63, 473-477. Menon, K.P.P. History of Kerala. Asian Educational Services, New Delhi, 1929; 1983 (reprint). (Cited from Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber, CRC Press, 2005.)

Peacock, V.E.; Kuneman, D.W. The inhibition of the formation of ,p-dimethylstyrene and p-cymene-8-ol in a carbonated citral-containing beverage system. J. Agric. Food Chem. 1985, 33, 330-335. (Reference from Liang et al., 2004.)

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Pruthy, J.S. Major spices of India: crop management, post-harvest technology. Indian Council of Agricultural Research, New Delhi, 1993, p. 514. (Cited from Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber, CRC Press, 2005.)

Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber. CRC Press, 2005.

Rosengarten, F.J. The Book of Spices. Livingston Pub. Co., PA, USA, 1969. (Cited from Ravindran, P.N.; Nirmal Babu, K. Ginger, The Genus Zingiber, CRC Press, 2005.)

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Schieberle, P.; Grosch, W. Potent odorants resulting from the peroxidation of lemon oil. Z Lebensm Unters Forsch. 1989, 189, 26-31.

Stoilova, I.; Krastanov, A.; Stoyanova, A.; Denev, P.; Gargova, S. Antioxidant activity of a ginger extract (Zingiber officinale). Food Chem. 2007, 102, 764-770.

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Thomas, A.F.; Bessiere, Y. Limonene. Nat. Prod. Rep. 1989, 6, 291-309.

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10. APPENDIX

Appendix 1: Chemical Composition of Ginger Oleoresins by GC (Alphabetical)

Whole Whole Buderim Pharma- Synthite Synthite Synthite Synthite Buderim Herb Herb Austra- Chem Component: Rajakumari Irutti Nigerian Shimoga Fijian Nigerian Indian lian Chinese

1,8-cineole 1.51 0.78 1.79 1.28 0.21 0.44 0.18 0.13 0.10 1-methyl-4-(1,5,9- trimethyl-4- 3.19 3.40 4.11 4.32 3.98 3.73 13.20 8.74 4.82 decenyl)-benzene alpha-amorphene 14.37 18.21 19.72 25.22 20.53 18.21 11.06 16.94 20.55

alpha-copaene 2.31 2.09 2.00 3.04 1.68 2.04 0.36 0.81 2.12 alpha-terpineol 1.86 2.00 3.37 2.94 3.35 1.33 1.78 2.82 2.70 alpha-zingiberene 266.05 255.95 160.27 167.40 137.44 226.6 18.90 25.55 198.44 5 ar-curcumene 83.19 65.42 67.90 137.57 88.41 109.8 75.61 101.12 73.32 2 aromadendrene 2.39 2.27 2.32 2.94 2.20 2.67 0.89 1.48 2.32 beta-bisabolene 62.52 60.46 46.18 61.62 50.70 64.33 25.32 33.75 55.28

beta-elemene 3.99 3.75 3.27 5.40 2.30 2.58 0.53 1.21 2.51 beta-elemol 3.55 3.66 3.58 4.51 3.14 1.95 3.57 2.96 2.41 beta-eudesmol 5.50 5.84 0.95 6.48 7.02 6.40 10.16 9.14 5.50 beta- 118.75 116.8 87.73 110.8 92.39 124.3 42.26 57.68 101.49 sesquiphellandrene beta- 17.47 16.90 19.61 16.68 22.84 26.21 26.21 19.63 19.10 sesquiphellandrol borneol 5.68 5.66 6.85 7.36 6.70 5.42 3.21 2.82 5.11 citronellol 1.68 1.83 1.58 1.96 1.15 0.44 2.67 2.42 1.93 decanal 1.15 1.57 1.37 2.94 2.10 0.98 0.89 0.94 1.06

gamma-bisabolene 4.97 6.10 6.01 6.38 6.39 4.53 3.57 5.65 4.82 geraniol 0.53 0.52 1.27 1.08 1.15 0.44 5.71 5.78 5.11 geranyl acetate 0.98 0.87 1.05 1.86 0.94 0.80 0.89 0.94 8.30 germacrene d 3.81 3.75 3.27 4.12 3.46 4.35 2.67 3.23 4.24 hexanal 0.71 0.87 1.05 2.06 1.36 0.44 0.89 1.34 0.87 linalool 4.97 4.09 1.69 1.96 1.15 3.38 0.53 0.54 0.96 sesquisabinene 7.72 6.45 5.06 7.56 6.91 5.15 8.20 6.72 6.27 hydrate trans,trans-alpha- 29.80 34.15 33.74 27.28 25.67 24.34 3.21 3.09 38.20 farnesene trans-beta- 4.97 4.88 3.58 5.40 3.56 3.55 0.71 0.94 3.86 farnesene trans-nerolidol 8.78 9.15 6.12 7.65 6.18 8.62 8.20 8.34 5.98 undecan-2-one 2.39 3.31 1.16 1.67 1.05 0.09 0.53 0.54 1.25 unknown compound 7.63 6.88 12.65 11.19 11.52 6.49 37.80 32.00 14.76 bp-137 mw-380 unknown compound 14.99 13.68 12.23 5.20 9.43 20.26 11.95 7.40 10.42 bp-171 mw-290 unknown sesquiterpene 5.68 6.36 7.49 7.36 8.28 8.53 9.27 6.86 6.08 compound bp-109 mw-238

Whole Whole Buderim Pharma- Components Synthite Synthite Synthite Synthite Buderim Herb Herb Austra- Chem Rajakumari Irutti Nigerian Shimoga Fijian continued: Nigerian Indian lian Chinese unknown sesquiterpene 10.46 11.33 8.75 11.19 9.43 7.37 9.45 8.07 8.97 compound bp-137 unknownmw-222 19.37 sesquiterpene 15.16 15.33 20.24 18.64 18.02 26.93 19.63 16.30 compound bp-69 mw-238

zingerone 9.93 10.98 8.86 11.28 10.79 7.11 10.88 9.68 9.16

zingiberenol 3.55 4.70 6.33 10.99 7.33 3.73 5.71 6.86 3.86 [10]-gingerdione 14.46 16.29 15.29 10.01 11.21 18.84 16.58 10.76 12.64

[10]-shogaol 25.81 29.71 37.22 30.62 42.11 35.01 77.03 53.25 36.66

[4]-gingerol (t) 1.86 2.18 4.11 1.96 4.71 1.33 2.85 3.36 3.18 [4]-shogaol 1.33 2.87 2.00 1.28 1.89 1.16 4.28 5.11 1.74 [6]- 2.75 2.61 3.80 3.24 3.25 1.78 12.84 5.78 2.80 dehydroshogaol 11.91 [6]-gingerdione 8.60 11.59 15.39 6.18 12.47 13.91 8.47 11.19

[6]-gingerol 109.61 112.03 174.19 118.83 173.69 82.63 123.40 160.68 132.16

[6]-gingerone 4.35 5.49 8.86 9.42 8.90 4.44 15.16 13.04 8.01 [6]-shogaol 65.36 77.01 116.09 79.68 116.49 78.01 279.07 267.98 102.16

[8]-gingerdione 6.47 4.70 9.17 5.40 5.97 7.73 8.02 4.71 6.37 [8]-gingerol (t) 11.26 10.19 14.87 10.21 13.41 10.31 10.34 9.01 12.35

[8]-shogaol 15.96 15.33 25.83 17.86 27.13 20.79 52.60 42.09 22.57

Total Adjusted Parts per 999.99 Thousand of 1000.01 1000.0 999.97 1000.0 999.99 999.98 999.99 1000.00

Named Components

Appendix 2: Chemical Composition of Ginger Oleoresins by GC (Retention Time)

Whole Whole Buderim Synthite Synthite Synthite Synthite Buderim PharmaChem Avg IE Components Herb Herb Austra- Rajakumari Irutti Nigerian Shimoga Fijian Chinese Nigerian Indian lian

3.87 hexanal 0.71 0.87 1.05 2.06 1.36 0.44 0.89 1.34 0.87

6.31 1,8-cineole 1.51 0.78 1.79 1.28 0.21 0.44 0.18 0.13 0.10

6.97 linalool 4.97 4.09 1.69 1.96 1.15 3.38 0.53 0.54 0.96

7.59 borneol 5.68 5.66 6.85 7.36 6.70 5.42 3.21 2.82 5.11 alpha- 7.83 1.86 2.00 3.37 2.94 3.35 1.33 1.78 2.82 2.70 terpineol 7.98 decanal 1.15 1.57 1.37 2.94 2.10 0.98 0.89 0.94 1.06

8.24 citronellol 1.68 1.83 1.58 1.96 1.15 0.44 2.67 2.42 1.93

8.48 geraniol 0.53 0.52 1.27 1.08 1.15 0.44 5.71 5.78 5.11 undecan-2- 8.84 2.39 3.31 1.16 1.67 1.05 0.09 0.53 0.54 1.25 one geranyl 9.76 0.98 0.87 1.05 1.86 0.94 0.80 0.89 0.94 8.30 acetate 9.85 alpha-copaene 2.31 2.09 2.00 3.04 1.68 2.04 0.36 0.81 2.12

9.98 beta-elemene 3.99 3.75 3.27 5.40 2.30 2.58 0.53 1.21 2.51 trans-beta- 10.62 4.97 4.88 3.58 5.40 3.56 3.55 0.71 0.94 3.86 farnesene aroma- 10.67 2.39 2.27 2.32 2.94 2.20 2.67 0.89 1.48 2.32 dendrene

10.83 ar-curcumene 83.19 65.42 67.90 137.6 88.41 109.8 75.61 101.1 73.32

10.90 germacrene d 3.81 3.75 3.27 4.12 3.46 4.35 2.67 3.23 4.24

10.99 alpha- 266.05 255.95 160.27 167.40 137.44 226.65 18.90 25.55 198.44 zingiberene 11.01 alpha- 14.37 18.21 19.72 25.22 20.53 18.21 11.06 16.94 20.55 amorphene 11.11 trans,trans- 29.80 34.15 33.74 27.28 25.67 24.34 3.21 3.09 38.20 alpha- 11.14 farnesenebeta- 62.52 60.46 46.18 61.62 50.70 64.33 25.32 33.75 55.28 bisabolene 11.28 beta- 118.75 116.82 87.73 110.78 92.39 124.30 42.26 57.68 101.49 sesquiphellan 11.35 gadrenemma - 4.97 6.10 6.01 6.38 6.39 4.53 3.57 5.65 4.82 bisabolene 11.44 beta-elemol 3.55 3.66 3.58 4.51 3.14 1.95 3.57 2.96 2.41

11.61 trans- 8.78 9.15 6.12 7.65 6.18 8.62 8.20 8.34 5.98 nerolidol 11.85 sesquisabinen 7.72 6.45 5.06 7.56 6.91 5.15 8.20 6.72 6.27 e hydrate 12.04 zingiberenol 3.55 4.70 6.33 10.99 7.33 3.73 5.71 6.86 3.86

12.09 zingerone 9.93 10.98 8.86 11.28 10.79 7.11 10.88 9.68 9.16

12.41 beta- 5.50 5.84 0.95 6.48 7.02 6.40 10.16 9.14 5.50 eudesmol 12.81 unknown 10.46 11.33 8.75 11.19 9.43 7.37 9.45 8.07 8.97 sesquiterpene 12.84 compoundbeta- bp- 17.47 16.90 19.61 16.68 22.84 26.21 26.21 19.63 19.10 sesquiphellan137 mw-222 13.86 unknowndrol 15.16 15.33 20.24 18.64 18.02 19.37 26.93 19.63 16.30 sesquiterpene 14.42 compoundunknown bp - 5.68 6.36 7.49 7.36 8.28 8.53 9.27 6.86 6.08 sesquiterpene69 mw-238 15.48 compound1-methyl- 4bp- - 3.19 3.40 4.11 4.32 3.98 3.73 13.20 8.74 4.82 109(1,5,9 mw--238 16.36 trimethyl[4]-shogaol-4- 1.33 2.87 2.00 1.28 1.89 1.16 4.28 5.11 1.74 decenyl)- [6]- 16.75 benzene 2.75 2.61 3.80 3.24 3.25 1.78 12.84 5.78 2.80 dehydroshoga 17.22 [4]-gingerolol 1.86 2.18 4.11 1.96 4.71 1.33 2.85 3.36 3.18 (t) 17.86 [6]-gingerone 4.35 5.49 8.86 9.42 8.90 4.44 15.16 13.04 8.01

18.38 [6]-shogaol 65.36 77.01 116.09 79.68 116.49 78.01 279.07 267.98 102.16

Whole Whole Buderim Avg IE Synthite Synthite Synthite Synthite Buderim PharmaChem Components Herb Herb Australia Cont’d Rajakumari Irutti Nigerian Shimoga Fijian Chinese Nigerian Indian n [6]- 18.66 8.60 11.59 15.39 6.18 12.47 11.91 13.91 8.47 11.19 gingerdione 19.15 [6]-gingerol 109.61 112.03 174.19 118.83 173.69 82.63 123.40 160.68 132.16

20.09 [8]-shogaol 15.96 15.33 25.83 17.86 27.13 20.79 52.60 42.09 22.57 unknown 20.30 compound bp- 7.63 6.88 12.65 11.19 11.52 6.49 37.80 32.00 14.76 137 mw-380 [8]- 20.35 6.47 4.70 9.17 5.40 5.97 7.73 8.02 4.71 6.37 gingerdione

unknown 20.71 compound bp- 14.99 13.68 12.23 5.20 9.43 20.26 11.95 7.40 10.42 171 mw-290

[8]-gingerol 20.78 11.26 10.19 14.87 10.21 13.41 10.31 10.34 9.01 12.35 (t)

21.58 [10]-shogaol 25.81 29.71 37.22 30.62 42.11 35.01 77.03 53.25 36.66 [10]- 21.81 14.46 16.29 15.29 10.01 11.21 18.84 16.58 10.76 12.64 gingerdione

986.7 Total: 986.89 987.64 980.99 987.83 981.87 988.35 987.62 984.47 9

Appendix 3. GC of Acetone Extract Synthite Rajakumari Ginger

Appendix 4. GC of Acetone Extract Synthite Irutti Ginger

Appendix 5. GC of Acetone Extract Synthite Nigerian Ginger

Appendix 6. GC of Acetone Extract Synthite Shimoga Ginger

Appendix 7. GC of Acetone Extract Whole Herb Nigerian Ginger

Appendix 8. GC of Acetone Extract Whole Herb Indian Ginger

Appendix 9. GC of Acetone Extract Buderim Australian Ginger

Appendix 10. GC of Acetone Extract Buderim Fijian Ginger

Appendix 11. GC of Acetone Extract PharmaChem Chinese Ginger

Appendix 12. HPLC of Hexane Extract Synthite Rajakumari Ginger

68.495 m in

64.255

63.265

62.817

61.317

60.384

59.319 60 58.851

57.027

55.601

54.621

53.195

52.420

51.683

51.347

50.329

49.688

49.363 50

48.519

(-)-Zingiberene 48.071

46.911

45.812

45.150

44.731

44.341

43.591

43.371

42.845 42.556

41.946

41.517

40.989

40.402

39.795

[10]-Shogaol 39.254

38.660

38.288 38.034

37.345

36.699 36.441

35.161 34.919

[8]-Shogaol 34.134

33.387

33.064

32.739

32.258

31.829

31.378

31.189 30.929

[10]-Gingerol 30.386

29.271 30

28.129

27.641

27.261

26.982

[6]-Shogaol 26.232 26.047

25.631

24.883

24.509

23.960

[8]-Gingerol 23.531 23.207

23.020 22.783

22.173

21.633

21.236

20.985

20.402

20.057

19.741

19.268 20

18.929

[6]-Gingerol 18.670

17.844

16.906

16.576

15.146

14.678

11.767

9.986

9.228 10

8.657

8.012

7.219

6.551

5.165

4.658 3.828 DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\SYNTHITE-19152-5UL.D) 0 0 500 m AU 2500 2000 1500 1000

Appendix 13. HPLC of Hexane Extract Synthite Irutti Ginger

69.471

m in 68.221

66.754

64.061

62.649

61.179

60.259

59.216 60

58.757

56.969

55.539

54.577

53.165

52.393

51.669

51.335

50.321

49.700

49.361 50

48.520

(-)-Zingiberene 48.075

46.922

45.825

45.162

44.745

44.354

43.606

43.370

42.859

42.572

41.959

41.523

40.985

40.397

[10]-Shogaol 39.795

39.247 40

38.656

38.272 38.033

37.351

36.696 36.437

35.158 34.927

34.141

[8]-Shogaol

33.398

33.075

32.746

32.273

31.846

[10]-Gingerol 31.392

31.205 30.942

30.395

29.292 30

28.815

28.138

27.656

27.278

[6]-Shogaol 26.995

26.094 25.948

25.656

24.899

24.520

23.975

23.538

[8]-Gingerol 23.230

23.033 22.798

22.177

21.647

21.242 21.011

20.420

20.059

19.767

19.288 20

18.922

[6]-Gingerol 18.681

17.873

16.895

16.578

15.158

11.797

9.994

8.679

7.975

7.214

6.522

5.183

4.605

3.800

DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\SYNTHITE-19153-5UL.D)

500

m AU

1000

1500

2000 2500

Appendix 14. HPLC of Hexane Extract Synthite Nigerian Ginger

m in 68.263

63.881

62.505

61.044

60.159

60 59.056

56.831

55.387

54.423

52.865

52.236

51.515

51.178

50.167

49.216 50

48.370

47.939

(-)-Zingiberene 47.585

46.795

45.699

45.041

44.624

44.240

43.710

43.471

43.299

42.739

42.449

41.841

41.433

40.910

40.371

39.719

39.179 40 [10]-Shogaol

38.592

38.223

37.980

37.736

37.281

36.647 36.400

35.679

35.137

34.886

[8]-Shogaol 34.108

33.362

33.044

32.723

32.239

31.809

31.357

31.161

[10]-Gingerol 30.912

30.372

30.079

29.248 30

28.091

27.620

27.239

[6]-Shogaol 26.964 26.549

26.348

25.905

25.614

24.848

24.476

23.949

23.507

23.183

[8]-Gingerol 22.985

22.749

22.189

21.602

21.219

20.951

20.670

20.399

20.039

19.740

19.266 20 18.913

18.652

[6]-Gingerol 18.159

17.824

17.417

16.892

16.563

15.134

14.660

13.978

13.540

12.454

11.756

10.991

9.978

9.217 10

8.626

8.178

7.204

5.677

5.167

4.787 4.572

4.239

3.760

DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\SYNTHITE-19154-5UL.D)

500

m AU

1000

1500

2000 2500

Appendix 15. HPLC of Hexane Extract Synthite Shimoga Ginger 69.000

m in

67.573

64.106

62.434

60.943 60.097

60 59.055

57.696

56.873

55.610

55.061

54.405

53.406

52.976

52.269

51.523

51.198

50.181

49.610

49.225 50

48.398

(-)-Zingiberene 47.962

46.810

45.711

45.059

44.651

44.255

43.494

43.323 43.085

42.763

42.477

41.864

41.524

40.929

40.597 40.357

39.725

39.183 40

38.610

[10]-Shogaol 37.979

37.171

36.832

36.649 36.383

35.683

35.132 34.867

[8]-Shogaol 34.090

33.344

33.042

32.726

[10]-Gingerol 32.272 31.795

31.496

31.151

30.911

30.350

29.922

29.242 30

28.128

27.613

27.225 26.969

26.249

[6]-Shogaol 25.882

24.856

24.499

23.965

23.526

23.199

22.737

[8]-Gingerol 22.499 22.208

21.615

21.236 20.964

20.054

19.738

19.275 20

18.932

[6]-Gingerol 18.648

18.170

17.822

17.602

17.431

16.904

16.590

15.652

15.145

14.672

13.988

13.547

12.950

12.587 12.439

11.776

10.006

9.236 10

8.618

8.312 8.028

7.204

5.157

4.419 3.688 DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\SYNTHITE-19155-5UL.D) 0 0 500 m AU 2500 2000 1500 1000

100

Appendix 16. HPLC of Hexane Extract Whole Herb Nigerian Ginger

m in

67.553

64.480

63.068

61.637

60.616

59.556 60

57.227

55.768

54.800

53.734

53.216

52.571

51.824

51.478

50.459

49.477 50

48.871 48.607

48.165

(-)-Zingiberene 47.825

46.978

45.884

45.210

44.803

44.391

43.864

43.609

43.424

42.883

42.586

41.976

41.578

41.049

40.714 40.499 39.841

[10]-Shogaol

39.285 40

38.687

38.078

37.371

36.744 36.477

35.216

34.981

[8]-Shogaol 34.185

33.445

33.117

32.811

32.329 31.890

[10]-Gingerol 31.225 30.979

30.432

29.323 30

28.823

28.165

[6]-Shogaol 27.681 27.304

26.942

26.122 25.966

25.648

24.904

24.537

24.012

23.543

23.262

23.040

22.837

[8]-Gingerol 22.587 [6]-Gingerol 22.247

21.663

21.278 21.035

20.447

20.070

19.806

19.327 20

18.937

18.709

18.364

17.910

17.640

17.458

16.905

16.594

15.842

15.193

14.726

14.053

13.579

11.872

11.105

10.083

9.419 10

8.938 8.701

8.379 8.113

7.358

6.932

6.632

5.231

4.188 3.930 0.292 DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\WHOLEHERB-NIGERIA-5UL.D) 0 0 500 m AU 2500 2000 1500 1000

101

Appendix 17. HPLC of Hexane Extract Whole Herb Indian Ginger

m in

66.379

64.340

62.942

61.460

60.547

59.438 60 58.997

57.184

55.686

54.716

53.690

53.248

52.517

51.771

51.413

50.416

49.784

49.424 50

48.799

48.545

(-)-Zingiberene 48.065

46.937

45.835

45.168

44.741

44.503

44.355

43.851

43.406

42.850

42.556

41.940

41.540

41.013

39.823

[10]-Shogaol 39.274 40

38.681

38.035

37.831

37.375

36.710 36.472

34.972

34.178

[8]-Shogaol 33.440

33.113

32.797

32.317

31.891

[10]-Gingerol 31.438 31.240

30.440

29.329 30

28.850

28.486

28.169

27.692

27.309

26.927

[6]-Shogaol 26.300

25.985

25.663

24.935

24.564

24.025

23.574

[8]-Gingerol 23.265 23.071

22.868

22.593

22.264

21.687

21.296

21.038

20.799

20.445

20.105

19.822

[6]-Gingerol 19.351 20

18.977

18.734

18.412

17.911

17.668

17.449

16.948

16.630

15.804

15.570

15.196

14.717

14.042

13.572

13.013

12.652 12.485

11.800

10.973

9.987

9.243 10

8.671

7.973

7.219

5.186

3.932

DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\WHOLEHERB-INDIA-5UL.D)

500

m AU

1000

1500

2000 2500

102

Appendix 18. HPLC of Hexane Extract Buderim Australian Ginger

m in

65.854

64.277

62.909

61.404

60.464

59.366 60

57.918

56.791

55.622

54.645

53.654

53.082

52.465

51.683

51.361

50.359

49.365 50

48.523

48.081

(-)-Zingiberene 47.341

46.898

46.503

45.804

45.143

44.745

44.322

43.787

43.549

43.360

42.813

42.530

42.301

41.920

41.534

40.998

40.701

40.458

40.224

39.810

39.263 40

38.676

38.061

[10]-Shogaol 37.487 37.269

36.750

36.490

36.122

35.244 35.000

34.411

34.009

33.480

32.844

[8]-Shogaol 32.371

31.927

31.625

31.276

31.026

30.480

30.179 [10]-Gingerol 30.014

29.350 30

28.808

28.220

27.726

[6]-Shogaol 27.339

26.940

26.432

25.999

25.679

24.914

24.573

[8]-Gingerol 24.053 23.635

23.285

22.899

22.280

21.700

21.302

20.993 20.786

20.471

20.101

19.817

19.337 20

[6]-Gingerol 18.949

18.712

18.229

17.873

16.908

16.615

15.845

15.172

12.631

11.762

10.984

9.937

9.182 10

8.614

7.923

7.177

5.157

4.716

3.870

DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\BUDERIM-AUSTRILIA-5UL.D)

500

m AU

1000

1500

2000 2500

103

Appendix 19. HPLC of Hexane Extract Buderim Fijian Ginger

m in

65.751

64.200

62.803

61.307

60.559

59.292 60

57.919

56.722

56.045

55.571

54.601

53.610

53.038

52.427

51.663

51.334

50.331

49.352 50

48.513

48.086

47.770

(-)-Zingiberene 46.901

46.516

45.815

45.157

44.755

44.338

43.798

43.404

42.843

42.549

42.326

41.939

41.603

40.983

40.687 40.531

39.799

39.256 40

[10]-Shogaol 38.663

38.049

37.460

37.233

36.735

36.462

36.095

35.751

35.202

[8]-Shogaol 34.956

34.372

34.178

33.960

33.428

33.095

32.792

32.324

[10]-Gingerol 31.871 31.564

31.216

30.975

30.432

30.152

29.296 30

28.753

28.158

27.682

27.297

26.897

[6]-Shogaol 26.316

25.957

25.653

24.872

24.538

[8]-Gingerol 24.015 23.568

23.242

22.842

22.553

22.249

21.649

21.268

20.983 20.709

20.424

20.063

[6]-Gingerol 19.777

19.297 20

18.922

18.676

18.180

17.829

17.412

16.875

16.553

15.765

15.128

14.655

13.973

13.506

11.753

10.993

9.955

9.209 10

8.641

5.160

3.855

DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\BUDERIM-FUJIAN-5UL.D)

500

m AU

1000

1500

2000 2500

104

Appendix 20. HPLC of Hexane Extract PharmaChem Chinese Ginger

m in

64.101

63.323

62.710

61.265

60.312

59.232 60

58.798

56.967

55.577

54.554

52.998

52.382

51.641

51.322

50.312

49.347 50

48.487

(-)-Zingiberene 48.068

46.903

45.807

45.144

44.739

44.338

43.787

43.565

43.382

42.837

42.548

41.938

41.515

40.989

40.436

39.794

39.252 40 [10]-Shogaol

38.660

38.043

37.807

37.361

36.715

36.464

36.114

35.190 34.950

34.167

[8]-Shogaol 33.422

33.105

32.778

32.302

31.869

[10]-Gingerol 31.215

30.429

30.096 29.954

29.309 30

28.803

28.158

27.682

[6]-Shogaol 27.302 27.015

26.307

25.962

25.656

24.910

24.537

[8]-Gingerol 24.014 23.562

23.247

22.831

22.254

21.657

21.270

21.007 20.772

20.450

20.074

19.786

19.304 20

18.942

[6]-Gingerol 18.688

17.850

17.427

16.897

16.590

15.146

14.672

13.989

13.529

11.769

10.977

9.980

9.233 10

8.666

8.211

7.228

5.169

4.714

3.864

DAD1 E, Sig=280,16 Ref=off (GINGER\110519KAB-HEXANE\PHARM-CHEM-5UL.D)

500

m AU

1000

1500

2000 2500

105

Appendix 21. HPLC of Acetone Extract Synthite Rajakumari Ginger

49.730

49.012 m in 48.689

47.696

47.128

46.717

45.913 45.482

(-)-Zingiberene 44.401

43.659

43.299

42.657

42.290

41.888

41.404

41.176

40.932

40.452

40.164

39.601 40 39.257

38.730

38.078

37.544

[10]-Shogaol 36.995

36.413

35.823

35.218

34.537

34.276

33.893

33.597

33.398

33.063

32.832 32.023

[8]-Shogaol 31.314

31.004

30.651

30.251

29.789

30 29.371

[10]-Gingerol 29.096

28.364

27.863

27.266

26.734

26.143

[6]-Shogaol 25.668

25.306

24.904

24.342

24.064

23.616

[8]-Gingerol 22.958

22.495

22.053

21.623

21.341

21.151

20.912

20.635

20.417

19.772

19.398 20

18.649

18.255

17.958

[6]-Gingerol 17.516

17.166

16.923

16.535

16.282

15.850 15.711

15.484

14.981

14.720

14.396

14.072

13.711

13.266

13.043

12.542

12.236

11.775

11.354

10.814

10.512

10.316

10.013 9.803

10 9.356

9.101

8.596

8.269

8.041

7.691

7.519

7.245

6.576

6.321

6.072

5.388

3.703

3.437

3.003 2.774 DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\SYNTHITE19152.D) 0 0 500 m AU 2500 2000 1500 1000

106

Appendix 22. HPLC of Acetone Extract Synthite Irutti Ginger

49.692

48.979 m in 48.657

47.663

47.099

46.691

45.896

45.462

(-)-Zingiberene

44.394 45

43.639

43.291

42.652

42.284

41.884

41.394

41.175

40.912

40.447

40.158

39.592 40 39.245

38.714

38.074

37.523

[10]-Shogaol 36.975

36.394

36.085

35.805

35.196

34.521 35 34.264

33.751

33.578

33.388

33.049

32.814

32.009

[8]-Shogaol

31.298

30.988

30.627

30.231

29.768

29.351

[10]-Gingerol 29.083

28.345

27.838

27.244

26.117

25.647

[6]-Shogaol 25.283

24.876

24.714

25 24.329

24.110

23.672

22.947

[8]-Gingerol 22.466

22.035

21.435

21.144

20.903

20.389

19.761

19.388 20

18.651

18.261

[6]-Gingerol 17.955

17.521

17.178

16.925

16.539

16.301

15.855

15.494

14.993

14.708

15 14.382

14.063

13.707

13.264

13.049

12.547

12.245

11.781

11.265

10.822

10.524

10.321

10.016

9.825

9.532 10 9.360

9.105

8.595

8.263

8.038

7.687

7.528

7.255

6.588

6.331

6.074

5.397 5.249

5.213

3.777

3.431

2.997

2.734

2.163

DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\SYNTHITE19153.D)

500

m AU

1000

1500

2000 2500

107

Appendix 23a. HPLC of Acetone Extract Synthite Nigerian Ginger

49.651

48.949 m in 48.621

47.641

47.037

46.679

45.875

45.453

(-)-Zingiberene

45 44.379

43.289

42.654

42.282

41.883

41.404

41.151 40.982

40.446

40.155

39.589 40 39.250

38.712

38.127

37.520

[10]-Shogaol 36.972

36.389

35.798

35.598

35.197

35.025

34.516 35 34.262

33.574

33.388

33.052

32.809

[8]-Shogaol 32.011

31.299

31.003

30.636

30.239

29.770

29.354

[10]-Gingerol 29.091

28.353

27.836

27.240

26.954

26.118

25.653

25.298

[6]-Shogaol 24.891

24.697

25 24.378

23.955

23.736

[8]-Gingerol 22.942

22.461

22.065

21.632

21.340

21.143

20.907

20.352

19.760

19.397 20

19.068

18.649

18.255

17.944

[6]-Gingerol 17.599

17.170

16.909

16.524

16.292

15.851

15.369

14.991

14.689

15 14.367

14.045

13.696

13.350 13.239

13.033

12.518

12.221

11.984

11.758

11.246

11.046

10.801

10.510

10.306

10.003

9.837

9.693

9.524 10 9.359

9.110

8.588

8.248

7.692

7.524

7.259

6.589

6.319

6.091

5.411

5.175

4.047

3.721

3.449

2.992

2.721

2.146

DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\SYNTHITE19154.D)

500

m AU

1000

1500

2000 2500

108

Appendix 23b. HPLC of Hexane Washed, Acetone Extract Synthite Nigerian Ginger

109

Appendix 24. HPLC of Acetone Extract Synthite Shimoga Ginger

49.534

48.786 m in

48.475

47.505

46.966

46.545

45.766 45.347

45 (-)-Zingiberene 44.301

43.206

42.587

42.226

41.830

41.350

40.953

40.706

40.553

40.402

40.117

39.555 40 39.221

38.679

38.314

38.049

37.484

37.235

36.940

[10]-Shogaol 36.390

35.788

34.964

34.502 35

34.238

33.884

33.602

33.055 32.777

[8]-Shogaol

31.995

31.283

30.642

30.245 29.751

[10]-Gingerol 29.341

29.095 28.902

28.335

27.877

27.241

26.122

25.653

[6]-Shogaol 25.288

25.017

24.720

25 24.178

23.706

22.946

[8]-Gingerol 22.567

22.079

21.453

21.184

20.902

20.340

19.764

19.389 20

18.258

[6]-Gingerol 17.939

17.599 17.506

17.169

16.905

16.631

16.332

15.753

15.539 15.366

14.905

14.668

14.395 15

14.203

14.052

13.681

13.273

13.041

12.535

12.222

11.789

11.406

11.255

10.813

10.526

10.027 9.870

10 9.366

9.105

8.588

8.265

8.013

7.515

7.163

6.317

5.384 5.185

3.748

3.463

3.260

3.007

2.839 2.752 2.144 DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\SYNTHITE19155.D) 0 0 500 m AU 2500 2000 1500 1000

110

Appendix 25. HPLC of Acetone Extract Whole Herb Nigerian Ginger

49.315

m in

48.647

48.338

47.374

46.897

46.438

45.833 45.677

45.245

44.944

(-)-Zingiberene

45 44.228

43.133

42.511

42.148

41.764

41.282

40.901

40.333

40.047

39.490 40

39.137

38.611

38.295

38.046

37.415

[10]-Shogaol 36.879

36.300

35.716

35.099 34.925

34.438 35 34.182

33.689

33.294

32.977

32.709

[8]-Shogaol 31.937

31.225

30.927

30.563

30.168

29.702

30 29.288

[10]-Gingerol 29.038

28.307

28.010

27.784

27.194

26.723

26.071

25.616

25.246

[6]-Shogaol 24.855

24.678 25

24.303

23.917

23.710

22.912

22.540

[8]-Gingerol 22.414

22.035

21.629

21.302

20.880

20.307

19.735

20 19.380

19.135

18.628

18.256

17.914

[6]-Gingerol 17.546

17.179

16.888

16.506

15.839

15.595

15.381

14.983

14.672 15 14.329

14.034

13.681

13.371 13.249

13.027

12.522

12.245

11.962 11.766

11.256

11.015

10.791

10.502

9.958

9.677 10 9.356

9.146

8.759

8.555

8.198 8.064

7.500

7.285

6.802

6.571

6.302

6.075

5.393

5.167

3.832

3.483

2.995

2.726

2.153

DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\WHOLEHERB-NIGERIA.D)

500

m AU

1000

1500

2000 2500

111

Appendix 26. HPLC of Acetone Extract Whole Herb Indian Ginger

49.456

m in 48.730

48.396

47.440

46.946

46.472

45.400

(-)-Zingiberene

45 44.275

43.517

43.149

42.532

42.173

41.928 41.790

41.326

40.925

40.356

40.069

39.505 40

39.155

38.627

38.023

37.433

36.896

36.325

36.021

35.731

35.510

35.124

34.448 35 [10]-Shogaol 34.208

33.707

33.323

32.749

32.228

[8]-Shogaol 31.962

31.254

30.952

30.577

30.183

29.720

30 29.309

[10]-Gingerol 29.056

28.323

28.045

27.797

27.217

26.096

25.636

25.272

[6]-Shogaol 25.036 24.875

24.687 25 24.319

23.717

[8]-Gingerol 22.940

22.435

22.023

21.651

21.325

21.127

20.907

20.411

19.771

19.396 20 19.185

18.647

18.272

17.933

[6]-Gingerol 17.542

17.191

16.912

16.519 16.396

15.845

15.590 15.405

15.016

14.672 15 14.336

14.040

13.688

13.506

13.242

13.030

12.526

12.245

11.755

11.435 11.253

10.790

10.502

10.273

9.987

9.674

9.535 10 9.358

8.754

8.563

8.218

7.996

7.499

7.228

6.783

6.556

6.300

6.055

5.409

5.152

3.861

3.490

3.388

3.008

2.738

DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\WHOLEHERB-INDIA.D)

500

m AU

1000

1500

2000 2500

112

Appendix 27. HPLC of Acetone Extract Buderim Australian Ginger

49.093

m in

48.653

48.367

47.427

46.902

46.467

45.699

45.287

44.616 45

44.242

(-)-Zingiberene 43.808

43.157

42.538

42.175

41.773

41.272

40.910

40.659

40.343

40.057

39.862

39.501 40

39.153

38.625

38.059 37.857

37.432

36.900

[10]-Shogaol

36.318

35.966

35.730

35.172

34.932

34.462 35 34.205

33.548

32.997

32.725

32.423

[8]-Shogaol 32.118 31.763

31.250

30.751

30.582

[10]-Gingerol 30.194

29.720

30 29.293

29.061

28.325

28.033 27.850

27.195

26.638

26.095

25.635

[6]-Shogaol 25.242

24.854

25 24.343

23.925

23.702

[8]-Gingerol 22.902

22.556

22.050

21.651

21.318

20.909

20.598

20.317

19.773

19.415 20

19.064

18.674

18.268

17.938

[6]-Gingerol

17.555

17.193

16.916

16.523

15.836

15.601

15.370

14.957

14.657 15 14.326

14.034

13.689

13.490

13.240 13.073

12.510

12.223

11.749

11.540

11.243

10.773

10.491

10.242

10.012 9.851

9.512 10 9.343

9.070

8.743

8.546

8.197

7.980

7.480

7.233

6.825

6.540

6.282

6.059

5.347

3.787

3.457

2.995

2.729

2.179

DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\BUDERIM AUSTRALIAN.D)

500

m AU

1000

1500

2000 2500

113

Appendix 28. HPLC of Acetone Extract Buderim Fijian Ginger

49.353

m in

48.662

48.378

47.417

46.472

45.714

45.274

44.993

44.241

43.847

(-)-Zingiberene 43.156

42.538

42.176

41.774

41.275

40.921

40.669

40.345

40.054

39.851

39.496 40 39.234

38.621

38.301 38.159

37.420

36.888

36.305

[10]-Shogaol 35.959 35.722

35.163

34.930

34.468

34.199

33.549

33.285

32.996

32.724

32.424

32.133

[8]-Shogaol 31.752

31.246

30.950

30.584

30.199

29.717

[10]-Gingerol 29.054

28.319

28.037

27.179

26.633

26.394

26.083

25.631

25.243

[6]-Shogaol 24.852

24.326

23.930

[8]-Gingerol

22.902

22.564

22.062

21.638

21.314

20.897

20.614

20.310

19.754

19.402 20

19.062

18.642

18.258

[6]-Gingerol 17.928

17.550

17.187

16.902

16.509

16.291

15.829

15.356

14.940

14.651

14.329

14.029

13.669

13.231 13.037

12.510

12.220

11.753

11.234

10.774

10.493

10.249

9.854

9.514 10 9.352

9.105

8.751

8.559

8.199

8.000

7.497

7.263

6.837

6.576

6.312

6.085

5.370 5.225

3.778

3.498

2.997

2.729

2.144

DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\BUDERIM FIJIAN.D)

500

m AU

1000

1500

2000 2500

114

Appendix 29. HPLC of Acetone Extract PharmaChem Chinese Ginger

49.464

m in 48.769

48.452

47.486

46.991

46.535

45.733

45.337

45 (-)-Zingiberene 44.299

43.765

43.547

43.189

42.558

42.202

41.807

41.311

41.079 40.932

40.380

40.087

39.528 40 39.187

38.652

38.067

37.456

[10]-Shogaol 36.914

36.332

36.025

35.747

35.529

35.142

34.468 35 34.217

33.745

33.335

33.009

32.754

[8]-Shogaol 31.970

31.258

30.959

30.577

30.201

29.731

30 [10]-Gingerol 29.315

29.063

28.329

27.837

27.217

26.674

26.100

25.638

[6]-Shogaol 25.272

24.871

24.686 25

24.329

23.933

23.718

[8]-Gingerol 22.918

22.447

22.047

21.617

21.317

20.893

20.317

19.749

19.391 20 19.200

18.650

18.257

[6]-Gingerol 17.935

17.597

17.186

16.912

16.526

15.850

15.560

15.375

14.962

14.689

15 14.354

14.049

13.695

13.378

13.251 13.084

12.529

12.264

11.975

11.764

11.256

11.045

10.788

10.502

10.259

9.844

9.677 10 9.345

9.129

8.723

8.555

8.204

8.012

7.647 7.502

7.268

6.798

6.556

6.302

5.390

5.229

3.872

3.358

2.995

2.726

2.148

DAD1 C, Sig=280,16 Ref=off (GINGER\110415SL\PHARMA CHEM.D)

500

m AU

1000

1500

2000 2500

115

Appendix 30: Peak Area Summation of Gingerols and Shogaols from HPLC

Whole Whole Pharma Synthite Synthite Synthite Synthite Herb Herb Buderim Buderim Chem Peak Area Rajakumari Irutti Nigerian Shimoga Nigerian Indian Australian Fijian Chinese Hexane Gingerol Area: 25026.75 22440.68 43529.70 44371.15 40651.50 34074.51 11576.48 31089.48 36202.09 Hexane Shogaol Area: 13372.09 11968.53 21048.17 13346.46 20820.40 18169.87 30446.72 37906.87 20272.00 Hexane Total Area: 38398.84 34409.21 64577.87 57717.60 61471.90 52244.38 42023.20 68996.35 56474.09

Acetone Gingerol Area: 64951.45 68293.89 74292.32 61574.47 69831.28 55195.54 34296.34 48399.82 67552.80 Acetone Shogaol Area: 18856.25 19849.65 25356.26 17988.23 23919.88 25050.72 34847.26 42712.35 25962.25 Acetone Total Area: 83807.70 88143.54 99648.58 79562.70 93751.15 80246.26 69143.60 91112.17 93515.06

Appendix 31. Hexane Fluorescence Decay Curve

ORAC Fluorescence Decay Curve for Hexanes Extractions

600000 Bud Australia Budarim 500000 Fijian WH India

400000 WH Nigeria Pharma 300000 Chem Synth Raj 52

200000Fluorescence Synth Irutti 53 Synth 100000 Nigeria 54 Synth Shimoga 0 55 0 4 8 11 15 18 22 25 29 33 37 40 44 48 53 56 61 65 Time (minutes)

116

Appendix 32. Acetone Fluorescence Decay Curve

ORAC Fluorescence Decay Curve for Acetone Extractions 500000 Bud Austr 450000 Bud Fij 400000 WH India 350000 WH 300000 Nigeria PC 250000 Chines e Synth 200000 Raj 52

150000Fluorescence Synth Irutti 53 100000 Synth Nig 54 50000 Synth Shim 0 55 0 4 8 11 15 18 22 25 29 33 37 40 44 48 53 56 61 65 Time (minutes)

Appendix 33. The LC/MS Chromatograms of F166 and F167

*F166, [6]-Gingerol ( top) and F167 (bottom)

117

Appendix 34. The LC/MS Chromatograms of F126, F111, F142 and F167

Y:\Xcalibur\...\F126-05%-5ul-newMeth 1/13/2012 10:52:47 PM

RT: 12.60 - 33.60 19.56 NL: 5.10E6 357.12708 100 Base Peak F: ITMS + c APCI corona Full ms 18.85 [50.00-1000.00] MS 387.06351 F126-05%-5ul-newMeth 50 20.59 249.07585 0 18.83 NL: 4.15E6 387.03937 100 Base Peak F: ITMS + c APCI corona Full ms 17.02 [50.00-1000.00] MS 389.13327 ginger-top5-f111-05%-10ul- 50 19.29 newmethod 373.06696 0 19.20 NL: 1.75E6 233.11540 100 Base Peak F: ITMS + c APCI 17.98 corona Full ms 341.11169 [50.00-1000.00] MS 19.92 f142-05%-10ul-newmethod 50 203.10791

0 25.72 NL: 4.11E6 431.07260 100 Base Peak F: ITMS + c APCI 26.14 corona Full ms 277.10434 [50.00-1000.00] MS 25.40 f167-05%-5ul-newmeth 50 431.05734

0 33.13 NL: 1.48E7 354.28436 100 Base Peak F: FTMS + c ESI 32.90 Full ms [100.00-1000.00] 16.61 27.78 30.34 MS 500767005-f313 15.20 19.71 24.11 167.01225 167.01237 50 167.01230 163.13234 167.01230 167.01225 167.01230

0 14 16 18 20 22 24 26 28 30 32 Time (min)

*F313 was not determined

118

Appendix 35. The LC/MS Chromatograms of F108, F110 and F165

y:\xcalibur\...\exact mass\500477605-108 5/15/2012 2:23:01 PM 108

RT: 13.13 - 30.57 18.01 NL: 1.69E9 373.16418 100 Base Peak F: FTMS + c APCI corona Full ms 80 [100.00-1000.00] MS 500477605-108 60 18.26 40 343.15396

RelativeAbundance 20 14.43 17.19 18.99 27.57 30.56 371.14859 391.17484 275.09116 22.30 24.35 26.43 181.08566 140.08159 140.08156 140.08156 140.08153 0 18.87 NL: 1.45E9 377.19553 Base Peak F: 100 19.17 FTMS + c APCI 377.19537 corona Full ms 80 [100.00-1000.00] MS 500477606-110 60 18.51 407.20575 40 20.56 18.15 RelativeAbundance 20 355.15375 15.01 389.15909 21.55 25.91 27.60 28.92 371.14835 193.08553 140.08156 140.08151 140.08156 0 27.33 NL: 1.74E9 277.17953 100 Base Peak F: 26.04 FTMS + c APCI 297.20578 corona Full ms 80 [100.00-1000.00] MS 500477607-165 60

RelativeAbundance 20 17.96 25.05 15.80 21.58 23.37 27.87 334.23703 261.18466 117.06548 115.08626 140.08151 275.16394 0 14 16 18 20 22 24 26 28 30 Time (min)

119

Appendix 36. The LC/MS and MS2 of F126 and the Commercial Standard (Hexahydrocurcumin) for Confirmation

Y:\Xcalibur\...\120703-APCI\gigner-F126 7/3/2012 3:22:36 PM

RT: 14.37 - 23.41 SM: 7B 19.57 NL: 4.99E6 357.21 Base Peak F: ITMS 100 + c APCI corona Full 80 ms [50.00-1000.00] 18.91 MS gigner-F126 60 387.22 F126 40 20.61 17.70 18.33 249.22 20 14.82 15.12 16.35 17.35 373.24 20.94 21.57 21.90 22.61 RelativeAbundance 385.26 355.23 419.20 511.17 179.17 163.15 208.17 163.12 163.16 19.57 NL: 4.97E6 357.27 Base Peak F: ITMS 100 + c APCI corona Full 80 ms [50.00-1000.00] hexahydrocurcumin MS 60 hexahydrocurcumin 40

20 14.95 15.48 15.74 16.45 17.33 17.82 18.25 19.24 20.18 20.63 21.20 22.30 23.28

RelativeAbundance 140.16 163.19 140.16 140.13 140.14 140.13 140.13 327.29 371.23 163.13 163.12 163.15 140.14

15 16 17 18 19 20 21 22 23 Time (min)

357.24 NL: 4.78E6 100 gigner-F126#2737 RT: 19.61 AV: 1 F: 80 F126 ITMS + c APCI corona 60 Full ms [50.00-1000.00] 40

20 177.17 RelativeAbundance 339.32 179.18 207.20 233.26 249.46 271.46 297.32 313.35 374.29 357.24 NL: 5.53E6 5000000 hexahydrocurcumin#25 hexahydrocurcumin 68 RT: 19.55 AV: 1 F: 4000000 ITMS + c APCI corona Full ms 3000000 [50.00-1000.00] 2000000

1000000 177.18 339.29 179.17 207.17 221.30 233.32 249.25 271.30 290.32 301.16 313.37 374.36

180 200 220 240 260 280 300 320 340 360 380 m/z

120

Appendix 37. 1H-NMR of Hexahydrocurcumin

121

Appendix 38. The LC/MS and MS2 of F167 and the Commercial Standard ([6]- Gingerol) for Confirmation

y:\xcalibur\...\6-gingerol-1k-5ul 7/3/2012 1:20:40 PM

RT: 23.59 - 27.91 SM: 7B 25.78 NL: 3.49E6 431.24 Base Peak F: ITMS 100 F167 + c APCI corona Full 80 ms [50.00-1000.00] MS ginger-f167-apci 26.07 60 25.45 277.25 431.22 40 27.42 20 23.67 24.21 24.38 24.80 25.08 26.51 27.07

RelativeAbundance 249.19 547.21 355.21 163.15 261.22 371.20 401.28 275.30 0 26.10 NL: 6.03E5 277.25 Base Peak F: ITMS 100 + c APCI corona Full 80 ms [50.00-1000.00] 6-gingerol MS 6-gingerol-1k-5ul 60

40 25.03 177.17 20 23.89 24.06 24.62 25.56 25.85 26.75 27.28 27.45 27.75

RelativeAbundance 140.12 140.17 140.18 140.16 140.16 165.20 140.15 140.19 223.17 0 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 Time (min)

277.27 NL: 1.80E6 100 ginger-f167-apci#3565 RT: 26.09 AV: 1 F: 80 F167 177.19 ITMS + c APCI corona 60 Full ms [50.00-1000.00] 40

20 179.17 RelativeAbundance 259.29 123.29 137.11 163.25 207.14 220.19 249.40 293.27 307.31 277.25 NL: 8.06E5 800000 6-gingerol-1k- 6-gingerol 5ul#3471 RT: 26.10 600000 AV: 1 F: ITMS + c APCI 177.17 corona Full ms 400000 [50.00-1000.00]

200000 179.18 259.28 127.24 137.16 163.16 207.22 220.19 249.34 295.16 307.36

80 100 120 140 160 180 200 220 240 260 280 300 m/z

122

Appendix 39. 1H-NMR of [6]-Gingerol

123

Appendix 40. The LC/MS and MS2 of F142 and the Synthetic Standard ([4]- Gingerdiol) for Confirmation

y:\xcalibur\...\ginger-f142-5k-pos 7/3/2012 2:01:19 PM

RT: 14.87 - 22.48 SM: 7B 19.33 NL: 1.48E6 233.27 Base Peak m/z= 100 232.50-233.50 F: ITMS + 80 c APCI corona Full ms F142 [50.00-1000.00] MS 60 ginger-f142-5k-pos

40 18.43 233.24 20 15.32 16.36 16.76 17.29 17.60 18.15 19.89 20.41 20.70 21.26 21.71

RelativeAbundance 233.31 233.30 233.35 233.31 233.35 233.34 233.22 233.28 233.23 233.26 233.30 19.34 NL: 4.94E5 233.24 Base Peak F: ITMS + c 100 18.43 APCI corona Full ms [50.00-1000.00] MS 80 233.22 4-gingerdiol-1k-5ul 60 4-gingerdiol 40 17.46 20.09 20 15.41 15.67 16.51 16.99 233.22 17.78 233.23 20.72 21.11 21.78 22.48

RelativeAbundance 140.12 164.15 140.18 140.15 140.16 140.17 140.15 140.16 287.28

15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 Time (min)

233.22 NL: 9.79E5 100 F142 ginger-f142-5k- pos#2910 RT: 19.25 80 AV: 1 F: ITMS + c APCI 60 269.07 corona Full ms [50.00-1000.00] 40 163.12

20 RelativeAbundance 137.15 166.28 219.21 237.24 251.24 125.16 152.26 191.22 209.18 292.31 303.35 233.24 NL: 6.83E5 4-gingerdiol-1k- 600000 5ul#2597 RT: 19.34 4-gingerdiol AV: 1 F: ITMS + c APCI 269.09 corona Full ms 400000 [50.00-1000.00] 163.15 200000 137.13 251.17 127.23 151.16 177.23 201.32 219.27 249.28 263.29 292.26 312.35

80 100 120 140 160 180 200 220 240 260 280 300 m/z

124

Appendix 41. 1H-NMR of [4]-Gingerdiol

125

Appendix 42. The LC/MS and MS2 of F110 and the Synthetic Standard (Octahydrocurcumin) for Confirmation

y:\xcalibur\...\130222\ginger-f110-apci 2/22/2013 5:58:41 PM

RT: 13.46 - 23.22 17.13 NL: 1.17E6 371.29 17.30 Base Peak F: ITMS + c 100 371.31 APCI corona Full ms 80 F110 [50.00-1000.00] MS ginger-f110-apci 60 17.99 19.36 377.11 355.29 40 16.89 18.31 15.39 19.58 20 13.85 14.22 15.74 373.24 355.28 20.39 21.06 21.43 22.28 22.74 447.16 371.21 Relative Relative Abundance 371.25 563.23 507.13 193.16 181.13 371.27 163.20 163.15 17.73 NL: 1.23E5 341.29 Base Peak m/z= 100 340.50-341.50 F: ITMS 80 + c APCI corona Full octahydrocurcumin ms [50.00-1000.00] 60 MS ohc-100 40 17.97 17.36 18.38 20 14.00 14.64 15.59 16.09 16.59 341.23 19.15 19.72 20.36 21.20 22.19 22.52 Relative Relative Abundance 341.40 341.34 341.31 341.34 341.29 341.28 341.27 341.25 341.33 341.31 341.28 341.33 341.29

14 15 16 17 18 19 20 21 22 23 Time (min)

341.30 NL: 9.57E5 100 F110 ginger-f110-apci#2246 80 RT: 17.72 AV: 1 F: ITMS + c APCI corona 60 377.02 Full ms [50.00-1000.00]

20 163.16

Relative Relative Abundance 137.13 217.23 285.29 309.37 400.29 431.31 477.33 513.07 537.36 583.30 635.12 0 341.28 NL: 1.22E5 120000 ohc-100#2126 RT: 100000 octahydrocurcumin 17.71 AV: 1 F: ITMS + c APCI corona Full ms 80000 [50.00-1000.00] 377.04 60000 40000 163.15 20000 137.12 217.24 131.19 279.36 309.21 400.30 431.31 477.28 513.04 543.50 615.36 651.27 0 100 150 200 250 300 350 400 450 500 550 600 650 m/z

126

Appendix 43. 1H-NMR of Octahydrocurcumin Pure Fraction 5A

127

Appendix 44. The LC/MS and MS2 of F165 and the Synthetic Standard ([6]- Gingerdiol) for Confirmation

y:\xcalibur\...\ginger-f165-5k-pos 2/22/2013 7:21:03 PM

RT: 20.03 - 29.51 26.12 NL: 3.82E6 277.23 m/z= 53.91-624.18 F: 100 ITMS + c APCI corona 80 Full ms [50.00-1000.00] 24.79 F16 MS ginger-f165-5k-pos 60 261.30 5 40 25.79 23.79 21.29 21.78 23.11 295.26 26.68 27.86 20 21.02 22.82 387.21 28.69 533.23 Relative Relative Abundance 140.16 533.18 140.17 431.19 275.23 140.17 140.15 0 24.80 NL: 8.62E4 261.29 Base Peak F: ITMS + c 100 23.80 APCI corona Full ms 261.26 80 [50.00-1000.00] MS 6- 6-gingerdiol-100 60 gingerdiol 40 25.26 20.46 22.78 20 21.51 22.06 22.97 24.54 261.23 26.50 26.88 27.59 27.91 28.89 137.11 261.25 Relative Relative Abundance 140.16 140.12 140.13 140.15 140.13 140.16 140.15 140.15 140.16 0 21 22 23 24 25 26 27 28 29 Time (min)

261.30 NL: 6.36E5 100 ginger-f165-5k-pos#3031 80 297.11 RT: 24.79 AV: 1 F: F16 ITMS + c APCI corona 60 Full ms [50.00-1000.00] 5 40 279.20 20 163.20 Relative Relative Abundance 137.14 177.15 191.21 229.36 320.35 351.34 377.31 397.34 415.35 0 261.30 NL: 7.83E4 6-gingerdiol-100#2998 297.09 RT: 24.82 AV: 1 F: 60000 6- ITMS + c APCI corona Full ms [50.00-1000.00] 40000 gingerdiol 279.19 20000 163.19 137.15 113.18 177.23 191.29 229.35 320.34 351.39 391.18 411.44 0 100 150 200 250 300 350 400 m/z

128

Appendix 45. 1H-NMR of [6]-Gingerdiol

129

Appendix 46. Optimizing Antioxidant Concentrations Using ORAC Assay

Testing 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol 4-Gingerol Levels: 0.001% 0.002% 0.004% 0.006% 0.008% 0.01% 0.02% 0.04% 0.06% 0.08% 0.1% 1% Net AUC: 1.7267 5.83064 14.7643 18.6958 18.7414 19.1499 18.9481 19.1819 18.9113 18.6449 18.9131 23.2986

Testing 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol 4-Shogaol Levels: 0.001% 0.002% 0.004% 0.006% 0.008% 0.01% 0.02% 0.04% 0.06% 0.08% 0.1% 1% Net AUC: 2.44959 6.7991 13.9186 17.7593 18.6654 18.6985 18.3476 18.5654 18.3312 18.7418 18.6494 19.8228

4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- Testing Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Levels: 0.001% 0.002% 0.004% 0.006% 0.008% 0.01% 0.02% 0.04% 0.06% 0.08% 0.1% 1% Net AUC: 1.83642 8.6049 14.6625 18.9771 18.8056 19.2331 19.0613 18.9735 18.8633 18.8811 18.8554 21.9124

Testing 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol 6-Gingerol Levels: 0.006% 0.008% 0.01% 0.02% 0.04% 0.06% 0.08% 0.1% 0.2% 0.3% 1% Net AUC: 13.7369 18.3366 18.4815 18.4733 18.4099 18.7275 18.851 18.7144 18.5071 18.7502 18.8139

S-6- S-6- S-6- S-6- Testing Gingerol Gingerol Gingerol Gingerol Levels: 0.008% 0.02% 0.1% 0.6% Net AUC: 16.4926 18.5079 23.6127 18.5701

6- 6- 6- 6- 6- 6- 6- 6- 6- 6- 6- Testing Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Gingerdiol Levels: 0.006% 0.008% 0.01% 0.02% 0.04% 0.06% 0.08% 0.1% 0.2% 0.3% 1% Net AUC: 17.0711 18.7756 18.6916 18.7365 18.7335 18.8092 18.6673 18.5859 18.5273 18.7873 19.2402

Testing THC THC THC THC THC THC THC THC THC THC THC Levels: 0.0001% 0.001% 0.002% 0.004% 0.006% 0.008% 0.01% 0.02% 0.04% 0.06% 0.08% Net AUC: -2.5653 3.15036 8.4178 13.0997 15.2232 15.3689 15.2297 15.0316 15.3452 15.0316 15.2133

Testing THC THC THC THC THC HHC HHC HHC HHC HHC Levels: 0.1% 0.3% 0.5% 0.8% 1% 0.0001% 0.001% 0.01% 0.1% 1% Net AUC: 15.2247 14.75 14.7461 14.634 16.0468 -3.8236 2.21764 15.3073 15.5896 17.9958

Testing OHC OHC OHC OHC OHC OHC OHC OHC OHC OHC OHC Levels: 0.0001% 0.001% 0.002% 0.004% 0.006% 0.008% 0.01% 0.02% 0.04% 0.06% 0.08% Net AUC: -1.7363 1.09186 7.2987 12.6366 14.9283 15.2887 15.2639 15.7648 15.4658 15.4144 15.5198

Testing OHC OHC OHC OHC OHC Levels: 0.1% 0.3% 0.5% 0.8% 1% Net AUC: 15.499 15.964 15.6885 16.3023 17.7887

Toco- Toco- Toco- Toco- Toco- Toco- Toco- Toco- Toco- Toco- Toco- Toco- Testing pherols pherols pherols pherols pherols pherols pherols pherols pherols pherols pherols pherols Levels: 0.00006% 0.0001% 0.0005% 0.001% 0.003% 0.005% 0.008% 0.01% 0.03% 0.05% 0.08% 0.1% Net AUC: -4.1111 -4.193 -4.4775 -4.441 -4.3605 -3.3452 -4.2552 -3.9621 -3.6099 -3.3168 -2.692 -2.7037

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Appendix 47. LCMS of Tetrahydrocurcumin

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Appendix 48. 1H-NMR of [4]-Gingerol

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Appendix 49. 1H-NMR of [4]-Shogaol

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Appendix 50. Data Related to First Set of Antioxidants in Lemon Oil

Anti- Dilution Amt. of Filled Target Actual Amt. of Oil Titrant Peroxide Aver- Antioxid oxidant Used Dilution to (g) Conc. Conc. (g) for PV Volume Value age P.V. ant Conc. (g) (ppm) (mL) in Bvg 4- 0.1% in 0.108 7.996 12.5 ppm 13.51 5.02 10.55 20.32 21.84 gingerol, EtOH A 4- 0.1% in 0.099 7.996 12.5 ppm 12.38 5.00 12.57 24.44 gingerol, EtOH B 4- 0.1% in 0.102 7.997 12.5 ppm 12.75 5.01 10.75 20.76 1.25ppb gingero EtOH l, C 4- 0.1% in 0.1 7.994 12.5 ppm 12.51 5.02 12.35 23.90 22.87 gingerdi EtOH ol, A 4- 0.1% in 0.1 7.998 12.5 ppm 12.50 5.01 11.75 22.75 1.25ppb gingerd EtOH iol, B 4- 0.1% in 0.106 7.998 12.5 ppm 13.25 5.04 11.42 21.96 gingerdi EtOH ol, C 4- 0.1% in 0.108 7.996 12.5 ppm 13.51 5.03 10.82 20.82 21.72 shogaol, EtOH A 4- 0.1% in 0.1 8.087 12.5 ppm 12.37 5.01 11.62 22.50 shogaol, EtOH B 4- 0.1% in 0.107 8.002 12.5 ppm 13.37 5.01 11.29 21.84 1.25ppb shogaol, EtOH C 6- 1% in 0.104 8.02 125 ppm 129.68 5.01 12.81 24.87 23.04 gingerol, EtOH A 6- 1% in 0.101 8.008 125 ppm 126.12 5.00 11.56 22.42 12.5ppb gingero EtOH l, B 6- 1% in 0.108 7.997 125 ppm 135.05 5.00 11.26 21.82 gingerol, EtOH C S-6- 0.1% in 1.002 8.177 125 ppm 122.54 5.18 17.64 33.38 34.79 gingerol, Acetone A S-6- 0.1% in 0.989 8.304 125 ppm 119.10 5.00 19.42 38.14 gingerol, Acetone B S-6- 0.1% in 1.007 8.129 125 ppm 123.88 5.01 16.81 32.85 gingerol, Acetone C 6- 1% in 0.064 8.03 75 ppm 79.70 5.01 11.90 23.05 21.40 12.5ppb gingerd EtOH iol, A 6- 1% in 0.06 8.008 75 ppm 74.93 5.00 12.00 23.30 gingerdi EtOH ol, B

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6- 1% in 0.065 7.995 75 ppm 81.30 5.00 9.28 17.86 gingerdi EtOH ol, C 6- 1% in 0.099 8.017 125 ppm 123.49 5.00 13.01 25.32 25.65 12.5ppb shogaol, EtOH A 6- 1% in 0.101 8.008 125 ppm 126.12 5.07 14.64 28.19 shogaol, EtOH B 6- 1% in 0.115 8.02 125 ppm 143.39 5.01 12.10 23.45 shogaol, EtOH C 8- 1% in 0.1 8.002 125 ppm 124.97 5.01 14.98 29.20 25.32 gingerol, EtOH A 8- 1% in 0.097 8.021 125 ppm 120.93 5.07 12.44 23.85 12.5ppb gingero EtOH l, B 8- 1% in 0.101 8.015 125 ppm 126.01 5.01 11.83 22.91 gingerol, EtOH C 8- 1% in 0.099 8.004 125 ppm 123.69 5.03 10.82 20.82 21.03 12.5ppb shogaol, EtOH A 8- 1% in 0.099 8.01 125 ppm 123.60 5.02 10.35 19.92 shogaol, EtOH B 8- 1% in 0.104 8.009 125 ppm 129.85 5.02 11.57 22.35 shogaol, EtOH C 10- 1% in 0.099 8.063 125 ppm 122.78 5.01 11.88 23.01 24.18 gingerol, EtOH A 10- 1% in 0.105 7.996 125 ppm 131.32 5.01 13.12 25.49 gingerol, EtOH B 10- 1% in 0.098 8.006 125 ppm 122.41 5.03 12.44 24.04 12.5ppb gingero EtOH l, C 10- 1% in 0.102 8.003 125 ppm 127.45 5.08 15.12 29.07 26.69 shogaol, EtOH A 10- 1% in 0.1 7.998 125 ppm 125.03 5.07 13.60 26.13 12.5ppb shogaol, EtOH B 10- 1% in 0.103 7.992 125 ppm 128.88 5.02 12.83 24.86 shogaol, EtOH C Tetrahy 1% in 0.044 7.992 50 ppm 55.06 5.00 9.13 17.56 18.68 drocurc EtOH umin, A Tetrahy 1% in 0.041 8 50 ppm 51.25 5.01 9.29 17.84 5ppb drocurc EtOH umin, B Tetrahy 1% in 0.046 8.006 50 ppm 57.46 5.14 10.95 20.62 drocurc EtOH umin, C

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Hexahyd 1% in 0.102 8.005 125 ppm 127.42 5.02 12.51 24.22 21.49 rocurcu Acetone min, A Hexahy 1% in 0.1 8.002 125 ppm 124.97 5.00 10.42 20.14 12.5ppb drocurc Acetone umin, B Hexahyd 1% in 0.101 8.009 125 ppm 126.11 5.01 10.42 20.10 rocurcu Acetone min, C Octahyd 0.1% in 0.214 8.004 25 ppm 26.74 5.08 15.03 28.90 29.49 2.5ppb rocurcu EtOH min, A Octahyd 0.1% in 0.204 7.994 25 ppm 25.52 5.04 14.25 27.58 rocurcu EtOH min, B Octahyd 0.1% in 0.203 8.02 25 ppm 25.31 5.03 16.44 31.99 rocurcu EtOH min, C 50% 10% in 0.04 8.044 500 ppm 497.27 5.00 6.81 12.92 15.26 25ppb Tocoph EtOH erols, A 50% 10% in 0.048 8.105 500 ppm 592.23 5.00 10.62 20.54 Tocophe EtOH rols, B 50% 10% in 0.041 8.015 500 ppm 511.54 5.02 6.53 12.31 Tocophe EtOH rols, C Blank, 8.07 5.03 7.07 13.35 Old Refriger Pipette ated, A Blank, 8.348 5.00 7.46 14.21 Old Refriger Pipette ated, B Blank, 8.084 5.04 7.12 13.43 Old Refriger Pipette ated, C Blank, 5.10 8.44 15.86 15.86 0 Refriger ated, D Blank,(New 8.136 5.00 6.50 12.30 Old pipette)Hot Box, Pipette A Blank, 8.004 5.00 11.19 21.68 19.10 0 Hot Box, B Blank, 8.089 5.02 8.64 16.51 Hot Box, C *Note1: Only those in bold were tasted. Note2: S-6-gingerol (with strikethrough) was not included in tastings because of elevated levels of ethanol. All other strikethroughs reflect the use of a broken pipette, therefore, the results were discarded.

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Appendix 51. Data Related to Second Set of Antioxidants in Lemon Oil

Anti- Dilution Amt. of Filled Target Actual Amt. of Oil Titrant Peroxide Aver-age oxidant Used Dilution to (g) Conc. Conc. (g) for PV Volume Value P.V. (g) (ppm) (mL)

4-gingerol, 1% in 0.078 7.995 A EtOH 0.01% 97.56 5.00 9.20 18.00 20.20 4-gingerol, 1% in 0.079 8.064 B EtOH 0.01% 97.97 5.01 11.90 23.35 4-gingerol, 1% in 0.083 8.005 C EtOH 0.01% 103.69 4.99 9.80 19.24 4- 1% in gingerdiol, 0.077 7.999 EtOH A 0.01% 96.26 5.00 11.60 22.80 24.59 4- 1% in gingerdiol, 0.081 8.02 EtOH B 0.01% 101.00 5.00 12.20 24.00 4- 1% in gingerdiol, 0.088 8.111 EtOH C 0.01% 108.49 5.00 13.69 26.98 4-shogaol, 1% in 0.08 8.001 A EtOH 0.01% 99.99 4.99 11.73 23.11 22.66 4-shogaol, 1% in 0.081 7.989 B EtOH 0.01% 101.39 5.00 11.27 22.14 4-shogaol, 1% in 0.082 8.092 C EtOH 0.01% 101.33 5.00 11.56 22.72 6-gingerol, 1% in 0.798 8.066 A EtOH 0.10% 989.34 5.01 20.19 39.90 38.78 6-gingerol, 1% in 0.829 8.214 B EtOH 0.10% 1009.25 5.01 22.17 43.85 6-gingerol, 1% in 0.853 8.12 C EtOH 0.10% 1050.49 5.11 16.85 32.58 6- 1% in gingerdiol, 0.48 8.008 EtOH A 0.06% 599.40 5.00 20.00 39.60 35.48 6- 1% in gingerdiol, 0.48 8.05 EtOH B 0.06% 596.27 5.00 17.42 34.44 6- 1% in gingerdiol, 0.477 8.151 EtOH C 0.06% 585.20 4.99 16.37 32.40 Tetrahydro 1% in curcumin, 0.317 7.997 EtOH A 0.04% 396.40 5.01 17.03 33.59 30.80 Tetrahydro 1% in curcumin, 0.326 8.026 EtOH B 0.04% 406.18 5.01 14.84 29.22 Tetrahydro 1% in curcumin, 0.322 7.997 EtOH C 0.04% 402.65 5.00 15.00 29.60 Octahydroc 1% in 0.158 7.996 urcumin, A EtOH 0.02% 197.60 5.01 11.15 21.86 Octahydroc 1% in 0.16 8.008 urcumin, B EtOH 0.02% 199.80 5.00 14.52 28.64 28.53 Octahydroc 1% in 0.166 8.151 urcumin, C EtOH 0.02% 203.66 5.06 14.58 28.42 10% in Tocopherol 0.158 7.995 s, A EtOH 0.10% 1976.24 5.00 13.47 26.54 25.96 10% in Tocopherol 0.161 8 s, B EtOH 0.10% 2012.50 4.99 12.18 24.01 10% in Tocopherol 0.16 7.991 s, C EtOH 0.10% 2002.25 5.01 13.89 27.33

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Appendix 52. Bar Chart to Display Significance of First Set of Lemon Oils

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Appendix 53. Bar Chart to Display Significance of Second Set of Lemon Oils

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Appendix 54. Bar Chart to Display Significance of Paired Comparisons

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Appendix 55. Peak Areas from GC of Degradation Markers for Each Lemon Oil

Argentinian Lemon Limonene p-Cymene Limonene -Terpineol Geranial Neral Carvone 1X oxide Original Starting Oil 0.19 66.34 0 0.02 2.02 1.17 0 Control Fridge A 0.32 66.47 0.01 0.02 1.97 1.13 0.03 Control Fridge B 0.32 66.48 0.01 0.02 1.97 1.13 0.03 Control Fridge C 0.32 66.51 0.01 0.02 1.94 1.11 0.03 Control Fridge D 0.28 66.52 0.01 0.02 1.96 1.13 0.03 Control Hot Box A 0.67 66.43 0.06 0.02 1.96 1.12 0.03 Control Hot Box B 0.81 66.43 0.06 0.02 1.94 1.11 0.03 Control Hot Box C 0.7 66.45 0.07 0.02 1.92 1.1 0.03 4-Gingerol A 0.61 66.1 0.04 0.02 1.91 1.11 0.03 4-Gingerol B 0.68 66.16 0.06 0.02 1.88 1.09 0.03 4-Gingerol C 0.57 66.19 0.04 0.02 1.88 1.1 0.02 4-Gingerdiol A 0.62 66.15 0.04 0.02 1.94 1.12 0.03 4-Gingerdiol B 0.55 66.17 0.04 0.02 1.9 1.11 0.02 4-Gingerdiol C 0.66 66.13 0.05 0.02 1.94 1.12 0.03 4-Shogaol A 0.64 66.18 0.04 0.02 1.85 1.08 0.03 4-Shogaol B 0.62 66.2 0.04 0.02 1.87 1.09 0.03 4-Shogaol C 0.68 66.22 0.05 0.02 1.86 1.09 0.03 6-Gingerol A 0.62 66.17 0.04 0.02 1.92 1.12 0.03 6-Gingerol B 0.53 66.15 0.04 0.02 1.92 1.12 0.02 6-Gingerol C 0.59 66.17 0.04 0.02 1.92 1.12 0.03 6-Gingerdiol A 0.7 66.23 0.06 0.02 1.97 1.14 0.03 6-Gingerdiol B 0.62 66.25 0.04 0.02 1.93 1.11 0.03 6-Gingerdiol C 0.65 66.27 0.04 0.02 1.95 1.12 0.03 6-Shogaol A 0.73 66.13 0.06 0.02 1.96 1.13 0.03 6-Shogaol B 0.75 66.14 0.06 0.02 1.86 1.09 0.03 6-Shogaol C 0.64 66.17 0.06 0.02 1.84 1.08 0.03 8-Gingerol A 0.66 66.16 0.06 0.02 1.88 1.1 0.03 8-Gingerol B 0.73 66.19 0.06 0.02 1.89 1.1 0.02 8-Gingerol C 0.66 66.17 0.06 0.02 1.87 1.09 0.02 8-Shogaol A 0.65 66.17 0.06 0.02 1.87 1.09 0.03 8-Shogaol B 0.58 66.17 0.04 0.02 1.87 1.09 0.02 8-Shogaol C 0.58 66.21 0.04 0.02 1.87 1.09 0.02 10-Gingerol A 0.62 66.16 0.04 0.02 1.9 1.1 0.02 10-Gingerol B 0.77 66.21 0.06 0.02 1.92 1.12 0.03 10-Gingerol C 0.76 66.18 0.06 0.02 1.87 1.09 0.03 10-Shogaol A 0.65 66.14 0.06 0.02 1.76 1.03 0.02 10-Shogaol B 0.63 66.22 0.04 0.02 1.76 1.03 0.02 10-Shogaol C 0.68 66.27 0.06 0.02 1.7 1 0.02

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Tetrahydrocurcumin A 0.78 66.33 0.06 0.02 1.85 1.07 0.03 Tetrahydrocurcumin B 0.73 66.34 0.06 0.02 1.86 1.08 0.03 Tetrahydrocurcumin C 0.77 66.31 0.06 0.02 1.84 1.07 0.03 Hexahydrocurcumin A 0.97 66.34 0.07 0.02 1.86 1.07 0.03 Hexahydrocurcumin B 0.76 66.53 0.06 0.02 1.79 1.04 0.03 Hexahydrocurcumin C 0.93 66.52 0.07 0.02 1.82 1.05 0.04 Octahydrocurcumin A 0.64 65.75 0.06 0.02 1.79 1.05 0.03 Octahydrocurcumin B 0.75 65.77 0.06 0.02 1.78 1.05 0.04 Octahydrocurcumin C 0.69 65.93 0.06 0.02 1.75 1.04 0.03 Tocopherols A 0.58 66.46 0.06 0.02 1.92 1.01 0.03 Tocopherols B 0.66 66.5 0.07 0.02 1.91 0.97 0.02 Tocopherols C 0.59 67.17 0 0 1.82 0.93 0 2: 4-Gingerol A 0.58 66.19 0.04 0.02 1.81 1.06 0.02 2: 4-Gingerol B 0.72 66.22 0.06 0.02 1.83 1.07 0.03 2: 4-Gingerol C 0.61 66.28 0.04 0.02 1.68 0.99 0.02 2: 4-Gingerdiol A 0.71 66.33 0.06 0.02 1.79 1.05 0.02 2: 4-Gingerdiol B 0.72 66.37 0.06 0.02 1.87 1.09 0.02 2: 4-Gingerdiol C 0.68 66.33 0.06 0.02 1.79 1.05 0.02 2: 4-Shogaol A 0.61 66.31 0.05 0.02 1.79 1.05 0.02 2: 4-Shogaol B 0.69 66.31 0.06 0.02 1.84 1.08 0.02 2: 4-Shogaol C 0.66 66.3 0.06 0.02 1.74 1.03 0.02 2: 6-Gingerol A 0.59 63.86 0.04 0.02 1.66 0.98 0.02 2: 6-Gingerol B 0.62 63.62 0.04 0.02 1.67 0.99 0.02 2: 6-Gingerol C 0.51 63.64 0.03 0.02 1.76 1.03 0.02 2: 6-Gingerdiol A 0.61 64.99 0.04 0.02 1.72 1.02 0.02 2: 6-Gingerdiol B 0.56 65.06 0.04 0.02 1.71 1.01 0.02 2: 6-Gingerdiol C 0.56 65.05 0.04 0.02 1.68 1 0.02 2: Tetra- hydrocurcumin A 0.64 65.39 0.04 0.02 1.73 1.02 0.02 2: Tetra- hydrocurcumin B 0.52 65.48 0.04 0.02 1.66 0.99 0.01 2: Tetra- hydrocurcumin C 0.55 65.42 0.04 0.02 1.76 1.04 0.02 2: Octa- hydrocurcumin A 0.54 66.04 0.04 0.02 1.71 1.02 0.01 2: Octahydrocurcumin B 0.53 66.09 0.04 0.02 1.72 1.02 0.01 2: Octahydrocurcumin C 0.54 65.94 0.05 0.02 1.77 1.05 0.02 2: Tocopherols A 0.52 66.04 0.04 0.02 2 1.08 0.03 2: Tocopherols B 0.47 65.97 0.04 0.02 1.81 1 0.02 2: Tocopherols C 0.5 65.99 0.04 0.02 1.76 0.97 0.02

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Appendix 56. GC of Original Starting Argentinian Lemon Oil