GINGER (Zingiber officinale):

ANTIOXIDANTS AND THEIR USE IN STABILIZING LEMON OIL

by

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

ii

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.

iii

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.

iv

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.

v

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

vi

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

vii

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

viii

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

ix

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

x

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

xi

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

xii

LIST OF SCHEMES

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

Scheme 2. The reduction of DPPH

xiii

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

xiv

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

xv

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

xvi

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

1

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).

2

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).

3

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

4

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

O

OH

Figure 1. S-[6]-gingerol

O

OH

O

Figure 2. [6]-shogaol

5

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

6

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.

7

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

8

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.

9

O

OH

O OH

O

OH

O

O

OH

OH OH

O

OH

OAc OAc

O

OH

O O

O

OH

O OH

O

OH

O

OH

O

O

OH

O

OH

OH OH

O

OH

OR1

OH

OAc OAc

OH

R2

OR1

O

OH

O O

O

OH

a: R1= CH3, R2= H (3R,5S)

b: R1= CH3, R2= OCH3 (3R, 5S)

c: R1=R2=H (3S,5S)

Gingerol-related

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

10

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.

11

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).

12

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.

13

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).

14

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

O

OH

O

O

thymol

limonene oxide

perillyl alcohol

carvone

eucarvone

limonene

a

b

c

d

e

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

15

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-dimethyl-

styrene, 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).

16

O

OH OH

O

OH

OOH

citral

+H+

p-menthadien-8-ols

H+

-H2O

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

auto-oxidation

p-methyl-acetophenone

p-cresol

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.

17

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.

18

3. HYPOTHESIS

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

lemon oil.

19

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

20

without antioxidants: one refrigerated and one thermally accelerated sample, to

determine how effective the antioxidants are at preventing the lemon oil from

degrading.

21

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

22

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

23

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

24

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

25

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

26

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.

27

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.

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 Solvent for the samples: Acetone

28

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

29

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

30

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).

31

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

32

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

OH

O

O OH

OH

O

OHOH

NaBH4

EtOH

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

33

(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

OH

OO

OH

O O

OH

OO

OH

OH OH

NiCl2, NaBH4

EtOH

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),

34

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],

35

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).

36

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

37

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

Sample

Synthite

Rajakumari

Synthite

Irutti

Synthite

Nigerian

Synthite

Shimoga

Whole

Herb

Nigerian

Whole

Herb

Indian

Buderim

Australian

Buderim

Fijian

Pharma-

Chem

Chinese

Average

Moisture

Content

11.63%

11.22%

10.14%

10.58%

8.88%

10.89%

8.97%

9.42%

11.45%

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.

38

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.

39

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.

40

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.

41

6.1.4. Extraction of Ginger Powders

6.1.4.1. Acetone Extracts Using Orbital Shaker

Orbital

Shaker

Acetone

Extract

Synthite

Rajakumari

Synthite

Irutti

Synthite

Nigerian

Synthite

Shimoga

Whole

Herb

Nigerian

Whole

Herb

Indian

Buderim

Australian

Buderim

Fijian

Pharma-

Chem

Chinese

Yield;

Percent

Yield

1.51g;

5.03%

1.43g;

4.77%

1.81g;

6.03%

1.64g;

5.47%

2.04g;

6.8%

1.59g;

5.3%

1.31g;

4.37%

1.5g;

5%

1.61g;

5.37%

Table 2. Yields of acetone extracts using orbital shaker

6.1.4.2. Hexane Extracts Using Speed Extractor

Speed

Extractor

Hexane

Extract

Synthite

Rajakumari

Synthite

Irutti

Synthite

Nigerian

Synthite

Shimoga

Whole

Herb

Nigerian

Whole

Herb

Indian

Buderim

Australian

Buderim

Fijian

Pharma-

Chem

Chinese

Starting

Material

30.68g

35.07g

28.46g

28.39g

30.21g

25.76g

44g

32.31g

30.74g

Yield;

Percent

Yield

1.09g;

3.55%

1.02g;

3.58%

1.08g;

3.79%

1.62g;

5.71%

1.19g;

3.94%

1.03g;

4%

0.84g;

1.91%

0.94g;

2.91%

1.12g;

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-

42

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.

43

Ranking of

Gingerols &

Shogaols

Synthite

Rajakumari

Synthite

Irutti

Synthite

Nigerian

Synthite

Shimoga

Whole

Herb

Nigerian

Whole

Herb

Indian

Buderim

Australian

Buderim

Fijian

Pharma-

Chem

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

44

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

Scavenging

Activity (%)

Synthite

Rajakumari

Synthite

Irutti

Synthite

Nigerian

Synthite

Shimoga

Whole

Herb

Nigerian

Whole

Herb

Indian

Buderim

Australian

Buderim

Fijian

Pharma-

Chem

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.

45

ORAC

Values

Synthite

Rajakumari

Synthite

Irutti

Synthite

Nigerian

Synthite

Shimoga

Whole

Herb

Nigerian

Whole

Herb

Indian

Buderim

Australian

Buderim

Fijian

Pharma-

Chem

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

Extracts

Peak Area

Values

Peak Area

Ranking

DPPH

Values

DPPH

Ranking

ORAC

Values

ORAC

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

Chinese

56474

5

41.5

6

1.73

6

Table 7. Compilation of hexane extracts: values and rankings

46

Acetone

Extracts

Peak Area

Values

Peak Area

Ranking

DPPH

Values

DPPH

Ranking

ORAC

Values

ORAC

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

Chinese

93515

3

59

2

3.72

4

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

47

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.

48

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.

49

Initial

Concentration

Added to Well

Final

Concentration

in Well

[6]-

Gingerol

[6]-

Shogaol

[10]-

Gingerol

[10]-

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).

50

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

OMe OMe

CH 2 CH 2 CH CH 2

OH

C CH 2

OHO

CH 2

OH

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

octanediol)

Pr-n

MeO

OH OH

HO

R S

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

decanone)

(CH 2 ) 4

OMe

Me

O OH

HO

S

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

OMe

MeO

OMe

CH 2 CH 2 CH CH 2

OH

CH CH 2

OHHO

CH 2

OH

51

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

OMe OMe

CH 2 CH 2 CH CH 2

OH

CH CH 2

OH

HO

CH 2

OH

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

(CH 2 ) 4

OMe

Me

OH OH

HO

S R

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

(CH 2 ) 4

OMe

Me

O OH

HO

S

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

OMe OMe

CH 2 CH 2 C CH 2

O

CH

HO

CH 2

OHHO

CH 2

OH

52

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

OMe

CH 2 CH 2 C CH 2

O

CH CH 2

OH

HO

CH 2

OH

OH

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

Fraction Number

Retention Time (min)

(M+H)+

APCI (ms/ms)+

Molecular 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,

53

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).

54

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.

Antioxidant: Concentration in Lemon Oil

(Set 1): Concentration in Lemon Oil

(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.

55

Set 1: Set 2:

Antioxidant:

Concentration of Antioxidant in Lemon Oil:

Peroxide Value of

Lemon Oil:

Concentration of Antioxidant in Lemon Oil:

Peroxide Value of

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

56

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.

57

Antioxidant N

Rows Mean Std Errors Multiple

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

Antioxidant N Rows Mean Std Errors Multiple

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

58

Antioxidant N

Rows Mean Std Errors Paired

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

59

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

60

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.

61

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).

62

Attribute Sample Mean Std 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).

63

Attribute Sample Mean Std 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.

64

Attribute Sample Mean Std 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).

65

Attribute Sample Mean Std 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.

66

Attribute Sample Mean Std 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).

67

Attribute Sample Mean Std 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).

68

Attribute Sample Mean Std 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).

69

Attribute Sample Mean Std 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).

70

Attribute Sample Mean Std 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).

71

Attribute Sample Mean Std 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.

72

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-

73

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).

74

Lemon Oil Sample

p-Cymene

Limonene Limonene

oxide -

Terpineol Geranial Neral Citral Carvone

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

75

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

the GC.

76

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

77

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.

78

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.

79

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83

10. APPENDIX

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

Component: Synthite

Rajakumari Synthite

Irutti Synthite Nigerian

Synthite Shimoga

Whole Herb

Nigerian

Whole Herb

Indian

Buderim Austra-

lian

Buderim Fijian

Pharma-Chem

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-

decenyl)-benzene 3.19 3.40 4.11 4.32 3.98 3.73 13.20 8.74 4.82

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

226.65

18.90 25.55 198.44

ar-curcumene 83.19 65.42 67.90 137.57

88.41 109.82

75.61 101.12

73.32

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-sesquiphellandrene

118.75 116.8 87.73 110.8 92.39 124.3 42.26 57.68 101.49

beta-sesquiphellandrol

17.47 16.90 19.61 16.68 22.84 26.21 26.21 19.63 19.10

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 hydrate

7.72 6.45 5.06 7.56 6.91 5.15 8.20 6.72 6.27

trans,trans-alpha-farnesene

29.80 34.15 33.74 27.28 25.67 24.34 3.21 3.09 38.20

trans-beta-farnesene

4.97 4.88 3.58 5.40 3.56 3.55 0.71 0.94 3.86

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 bp-137 mw-380

7.63 6.88 12.65 11.19 11.52 6.49 37.80 32.00 14.76

unknown compound bp-171 mw-290

14.99 13.68 12.23 5.20 9.43 20.26 11.95 7.40 10.42

unknown sesquiterpene

compound bp-109 mw-238

5.68 6.36 7.49 7.36 8.28 8.53 9.27 6.86 6.08

84

Components continued:

Synthite Rajakumari

Synthite Irutti

Synthite Nigerian

Synthite Shimoga

Whole Herb

Nigerian

Whole Herb

Indian

Buderim Austra-

lian

Buderim Fijian

Pharma-Chem

Chinese

unknown sesquiterpene

compound bp-137 mw-222

10.46 11.33 8.75 11.19 9.43 7.37 9.45 8.07 8.97

unknown sesquiterpene

compound bp-69 mw-238

15.16 15.33 20.24 18.64 18.02 19.37

26.93 19.63 16.30

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]-dehydroshogaol

2.75 2.61 3.80 3.24 3.25 1.78 12.84 5.78 2.80

[6]-gingerdione 8.60 11.59 15.39 6.18 12.47 11.91

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

Thousand of Named

Components

1000.01 1000.0 999.97 1000.0 999.99 999.99

999.98 999.99 1000.00

85

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

Avg IE

Components

Synthite Rajakumari

Synthite Irutti

Synthite Nigerian

Synthite Shimoga

Whole Herb

Nigerian

Whole Herb

Indian

Buderim Austra-

lian

Buderim Fijian

PharmaChem Chinese

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

7.83 alpha-

terpineol 1.86 2.00 3.37 2.94 3.35 1.33 1.78 2.82 2.70

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

8.84 undecan-2-

one 2.39 3.31 1.16 1.67 1.05 0.09 0.53 0.54 1.25

9.76 geranyl acetate

0.98 0.87 1.05 1.86 0.94 0.80 0.89 0.94 8.30

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

10.62 trans-beta-farnesene

4.97 4.88 3.58 5.40 3.56 3.55 0.71 0.94 3.86

10.67 aroma-

dendrene 2.39 2.27 2.32 2.94 2.20 2.67 0.89 1.48 2.32

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-zingiberene

266.05 255.95 160.27 167.40 137.44 226.65

18.90 25.55 198.44

11.01 alpha-amorphene

14.37 18.21 19.72 25.22 20.53 18.21 11.06 16.94 20.55

11.11 trans,trans-alpha-

farnesene

29.80 34.15 33.74 27.28 25.67 24.34 3.21 3.09 38.20

11.14 beta-bisabolene

62.52 60.46 46.18 61.62 50.70 64.33 25.32 33.75 55.28

11.28 beta-sesquiphellan

drene

118.75 116.82 87.73 110.78 92.39 124.30

42.26 57.68 101.49

11.35 gamma-bisabolene

4.97 6.10 6.01 6.38 6.39 4.53 3.57 5.65 4.82

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-nerolidol

8.78 9.15 6.12 7.65 6.18 8.62 8.20 8.34 5.98

11.85 sesquisabinene hydrate

7.72 6.45 5.06 7.56 6.91 5.15 8.20 6.72 6.27

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-eudesmol

5.50 5.84 0.95 6.48 7.02 6.40 10.16 9.14 5.50

12.81 unknown sesquiterpene compound bp-137 mw-222

10.46 11.33 8.75 11.19 9.43 7.37 9.45 8.07 8.97

12.84 beta-sesquiphellan

drol

17.47 16.90 19.61 16.68 22.84 26.21 26.21 19.63 19.10

13.86 unknown sesquiterpene compound bp-

69 mw-238

15.16 15.33 20.24 18.64 18.02 19.37 26.93 19.63 16.30

14.42 unknown sesquiterpene compound bp-109 mw-238

5.68 6.36 7.49 7.36 8.28 8.53 9.27 6.86 6.08

15.48 1-methyl-4-(1,5,9-

trimethyl-4-decenyl)-benzene

3.19 3.40 4.11 4.32 3.98 3.73 13.20 8.74 4.82

16.36 [4]-shogaol 1.33 2.87 2.00 1.28 1.89 1.16 4.28 5.11 1.74

16.75 [6]-dehydroshoga

ol

2.75 2.61 3.80 3.24 3.25 1.78 12.84 5.78 2.80

17.22 [4]-gingerol (t)

1.86 2.18 4.11 1.96 4.71 1.33 2.85 3.36 3.18

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

86

Avg IE Cont’d

Components Synthite

Rajakumari Synthite

Irutti Synthite Nigerian

Synthite Shimoga

Whole Herb

Nigerian

Whole Herb

Indian

Buderim Australia

n

Buderim Fijian

PharmaChem Chinese

18.66 [6]-

gingerdione 8.60 11.59 15.39 6.18 12.47 11.91 13.91 8.47 11.19

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

20.30 unknown

compound bp-137 mw-380

7.63 6.88 12.65 11.19 11.52 6.49 37.80 32.00 14.76

20.35 [8]-

gingerdione 6.47 4.70 9.17 5.40 5.97 7.73 8.02 4.71 6.37

20.71 unknown

compound bp-171 mw-290

14.99 13.68 12.23 5.20 9.43 20.26 11.95 7.40 10.42

20.78 [8]-gingerol

(t) 11.26 10.19 14.87 10.21 13.41 10.31 10.34 9.01 12.35

21.58 [10]-shogaol 25.81 29.71 37.22 30.62 42.11 35.01 77.03 53.25 36.66

21.81 [10]-

gingerdione 14.46 16.29 15.29 10.01 11.21 18.84 16.58 10.76 12.64

Total: 986.89 987.64 980.99 987.83 981.87 988.35 986.7

9 987.62 984.47

87

Appendix 3. GC of Acetone Extract Synthite Rajakumari Ginger

88

Appendix 4. GC of Acetone Extract Synthite Irutti Ginger

89

Appendix 5. GC of Acetone Extract Synthite Nigerian Ginger

90

Appendix 6. GC of Acetone Extract Synthite Shimoga Ginger

91

Appendix 7. GC of Acetone Extract Whole Herb Nigerian Ginger

92

Appendix 8. GC of Acetone Extract Whole Herb Indian Ginger

93

Appendix 9. GC of Acetone Extract Buderim Australian Ginger

94

Appendix 10. GC of Acetone Extract Buderim Fijian Ginger

95

Appendix 11. GC of Acetone Extract PharmaChem Chinese Ginger

96

Appendix 12. HPLC of Hexane Extract Synthite Rajakumari Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E, S

ig=

28

0,1

6 R

ef=

off

(G

ING

ER

\11

05

19

KA

B-H

EX

AN

E\S

YN

TH

ITE

-19

15

2-5

UL

.D)

3.828

4.658 5.165

6.551 7.219

8.012 8.657

9.228

9.986

11.767

14.678 15.146

16.576 16.906

17.844

18.670 18.929 19.268 19.741 20.057

20.402 20.985 21.236

21.633 22.173 22.783 23.020 23.207 23.531 23.960 24.509

24.883 25.631 26.047 26.232 26.982 27.261

27.641 28.129

29.271

30.386 30.929 31.189 31.378

31.829 32.258

32.739 33.064

33.387 34.134

34.919 35.161

36.441 36.699 37.345 38.034 38.288

38.660 39.254

39.795 40.402

40.989 41.517

41.946 42.556 42.845 43.371 43.591

44.341 44.731

45.150 45.812

46.911

48.071 48.519

49.363 49.688

50.329

51.347 51.683

52.420

53.195

54.621

55.601

57.027

58.851 59.319

60.384

61.317

62.817 63.265

64.255

68.495

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol [8

]-G

ing

ero

l

[10

]-G

ing

ero

l

[6]-

Sh

og

ao

l

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

97

Appendix 13. HPLC of Hexane Extract Synthite Irutti Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\SY

NT

HIT

E-1

91

53

-5U

L.D

)

3.800

4.605 5.183

6.522 7.214

7.975 8.679

9.994

11.797

15.158

16.578 16.895

17.873

18.681 18.922 19.288 19.767 20.059 20.420 21.011 21.242

21.647 22.177 22.798 23.033 23.230 23.538 23.975 24.520

24.899

25.656 25.948 26.094

26.995 27.278 27.656

28.138 28.815

29.292

30.395 30.942 31.205 31.392

31.846 32.273

32.746 33.075

33.398 34.141

34.927 35.158

36.437 36.696 37.351 38.033 38.272

38.656 39.247

39.795 40.397

40.985 41.523

41.959 42.572 42.859 43.370 43.606

44.354 44.745

45.162 45.825

46.922

48.075 48.520

49.361 49.700

50.321

51.335 51.669

52.393

53.165

54.577

55.539

56.969

58.757 59.216

60.259

61.179

62.649

64.061

66.754

68.221

69.471

[10

]-S

ho

ga

ol

[8]-

Sh

og

ao

l

[6]-

Sh

og

ao

l

[6]-

Gin

ge

rol [8

]-G

ing

ero

l

[10

]-G

ing

ero

l

(-)-

Zin

gib

ere

ne

98

Appendix 14. HPLC of Hexane Extract Synthite Nigerian Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\SY

NT

HIT

E-1

91

54

-5U

L.D

)

3.760 4.239 4.572 4.787

5.167 5.677

7.204

8.178 8.626

9.217

9.978

10.991

11.756 12.454

13.540 13.978 14.660

15.134

16.563 16.892 17.417 17.824 18.159

18.652 18.913 19.266 19.740 20.039

20.399 20.670 20.951 21.219 21.602

22.189 22.749 22.985 23.183 23.507 23.949 24.476

24.848

25.614 25.905 26.348 26.549 26.964 27.239

27.620 28.091

29.248

30.079 30.372 30.912 31.161 31.357

31.809 32.239

32.723 33.044

33.362 34.108

34.886 35.137 35.679

36.400 36.647 37.281 37.736 37.980 38.223

38.592 39.179

39.719 40.371

40.910 41.433

41.841 42.449 42.739 43.299 43.471 43.710

44.240 44.624

45.041 45.699

46.795

47.585 47.939 48.370

49.216

50.167

51.178 51.515

52.236 52.865

54.423

55.387

56.831

59.056

60.159

61.044

62.505

63.881

68.263

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol [8

]-G

ing

ero

l

[10

]-G

ing

ero

l

[10

]-S

ho

ga

ol

[8]-

Sh

og

ao

l

[6]-

Sh

og

ao

l

99

Appendix 15. HPLC of Hexane Extract Synthite Shimoga Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\SY

NT

HIT

E-1

91

55

-5U

L.D

)

3.688 4.419

5.157

7.204

8.028 8.312 8.618

9.236

10.006

11.776 12.439 12.587 12.950 13.547 13.988 14.672

15.145 15.652

16.590 16.904 17.431 17.602 17.822 18.170 18.648 18.932 19.275 19.738

20.054

20.964 21.236 21.615

22.208 22.499 22.737 23.199 23.526 23.965 24.499

24.856

25.882 26.249

26.969 27.225 27.613

28.128

29.242 29.922 30.350 30.911 31.151 31.496 31.795 32.272

32.726 33.042 33.344

34.090

34.867 35.132 35.683

36.383 36.649 36.832 37.171

37.979 38.610

39.183 39.725

40.357 40.597 40.929

41.524 41.864

42.477 42.763 43.085 43.323 43.494

44.255 44.651

45.059 45.711

46.810

47.962 48.398

49.225 49.610 50.181

51.198 51.523 52.269 52.976 53.406

54.405 55.061 55.610

56.873

57.696

59.055

60.097

60.943

62.434

64.106

67.573

69.000

[6]-

Sh

og

ao

l

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

[10

]-G

ing

ero

l

[8]-

Gin

ge

rol

[6]-

Gin

ge

rol

(-)-

Zin

gib

ere

ne

100

Appendix 16. HPLC of Hexane Extract Whole Herb Nigerian Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\WH

OL

EH

ER

B-N

IGE

RIA

-5U

L.D

)

0.292

3.930 4.188

5.231

6.632 6.932 7.358 8.113 8.379 8.701 8.938 9.419

10.083

11.105

11.872

13.579 14.053 14.726

15.193 15.842 16.594 16.905 17.458 17.640 17.910 18.364 18.709 18.937

19.327 19.806 20.070 20.447 21.035 21.278

21.663 22.247 22.587 22.837 23.040 23.262 23.543 24.012 24.537

24.904 25.648 25.966 26.122

26.942 27.304

27.681 28.165 28.823

29.323

30.432 30.979 31.225

31.890 32.329

32.811 33.117 33.445

34.185

34.981 35.216

36.477 36.744 37.371 38.078

38.687 39.285

39.841 40.499 40.714

41.049 41.578

41.976 42.586

42.883 43.424 43.609 43.864

44.391 44.803

45.210 45.884

46.978

47.825 48.165 48.607 48.871

49.477

50.459

51.478 51.824

52.571 53.216

53.734

54.800

55.768

57.227

59.556

60.616

61.637

63.068

64.480

67.553

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l

[6]-

Sh

og

ao

l

[8]-

Sh

og

ao

l[1

0]-

Sh

og

ao

l

101

Appendix 17. HPLC of Hexane Extract Whole Herb Indian Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\WH

OL

EH

ER

B-I

ND

IA-5

UL

.D)

3.932

5.186

7.219 7.973

8.671 9.243 9.987

10.973

11.800 12.485 12.652 13.013 13.572 14.042

14.717 15.196

15.570 15.804

16.630 16.948 17.449 17.668 17.911 18.412 18.734 18.977 19.351 19.822 20.105

20.445 20.799 21.038 21.296

21.687 22.264 22.593 22.868 23.071 23.265 23.574 24.025 24.564

24.935 25.663 25.985 26.300 26.927 27.309

27.692 28.169 28.486 28.850

29.329

30.440

31.240 31.438 31.891

32.317 32.797 33.113

33.440 34.178

34.972

36.472 36.710 37.375

37.831 38.035 38.681

39.274 39.823

41.013 41.540

41.940 42.556 42.850

43.406 43.851 44.355 44.503 44.741

45.168 45.835

46.937

48.065 48.545 48.799 49.424 49.784 50.416

51.413 51.771

52.517 53.248 53.690

54.716

55.686

57.184

58.997 59.438

60.547

61.460

62.942

64.340

66.379

[10

]-S

ho

ga

ol

[8]-

Sh

og

ao

l

[6]-

Sh

og

ao

l

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l

(-)-

Zin

gib

ere

ne

102

Appendix 18. HPLC of Hexane Extract Buderim Australian Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\BU

DE

RIM

-AU

ST

RIL

IA-5

UL

.D)

3.870

4.716 5.157

7.177 7.923

8.614 9.182 9.937

10.984

11.762

12.631

15.172 15.845

16.615 16.908

17.873 18.229 18.712 18.949

19.337 19.817 20.101 20.471 20.786 20.993 21.302

21.700 22.280 22.899 23.285

23.635 24.053

24.573 24.914

25.679 25.999 26.432

26.940 27.339 27.726

28.220 28.808

29.350 30.014 30.179 30.480 31.026 31.276 31.625 31.927 32.371

32.844 33.480

34.009 34.411

35.000 35.244

36.122 36.490 36.750

37.269 37.487 38.061

38.676 39.263

39.810 40.224 40.458 40.701 40.998 41.534

41.920 42.301 42.530 42.813

43.360 43.549 43.787 44.322

44.745 45.143

45.804 46.503

46.898 47.341 48.081 48.523

49.365

50.359

51.361 51.683

52.465 53.082 53.654

54.645

55.622

56.791

57.918

59.366

60.464

61.404

62.909

64.277

65.854

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol [8

]-G

ing

ero

l[1

0]-

Gin

ge

rol

[6]-

Sh

og

ao

l [8]-

Sh

og

ao

l[10

]-S

ho

ga

ol

103

Appendix 19. HPLC of Hexane Extract Buderim Fijian Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\BU

DE

RIM

-FU

JIA

N-5

UL

.D)

3.855

5.160

8.641 9.209

9.955

10.993

11.753

13.506 13.973 14.655

15.128 15.765

16.553 16.875 17.412 17.829 18.180 18.676 18.922

19.297 19.777 20.063

20.424 20.709 20.983 21.268 21.649

22.249 22.553 22.842

23.242 23.568 24.015 24.538

24.872

25.653 25.957 26.316

26.897 27.297

27.682 28.158

28.753 29.296

30.152 30.432 30.975 31.216 31.564

31.871 32.324

32.792 33.095

33.428 33.960 34.178 34.372

34.956 35.202 35.751

36.095 36.462 36.735

37.233 37.460 38.049

38.663 39.256

39.799 40.531 40.687 40.983 41.603

41.939 42.326 42.549 42.843

43.404 43.798

44.338 44.755

45.157 45.815

46.516 46.901

47.770 48.086 48.513

49.352

50.331

51.334 51.663

52.427 53.038 53.610

54.601

55.571 56.045 56.722

57.919

59.292

60.559 61.307

62.803

64.200

65.751

[6]-

Sh

og

ao

l

[8]-

Sh

og

ao

l[1

0]-

Sh

og

ao

l

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l

104

Appendix 20. HPLC of Hexane Extract PharmaChem Chinese Ginger

min

01

02

03

04

05

06

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

E,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

51

9K

AB

-HE

XA

NE

\PH

AR

M-C

HE

M-5

UL

.D)

3.864

4.714 5.169

7.228

8.211 8.666

9.233 9.980

10.977

11.769

13.529 13.989 14.672 15.146

16.590 16.897 17.427 17.850

18.688 18.942 19.304 19.786 20.074

20.450 20.772 21.007 21.270

21.657 22.254 22.831 23.247

23.562 24.014 24.537

24.910 25.656 25.962 26.307 27.015 27.302

27.682 28.158

28.803 29.309

29.954 30.096 30.429

31.215 31.869 32.302

32.778 33.105

33.422 34.167

34.950 35.190

36.114 36.464 36.715

37.361 37.807 38.043

38.660 39.252

39.794 40.436

40.989 41.515

41.938 42.548 42.837 43.382 43.565 43.787

44.338 44.739

45.144 45.807

46.903

48.068 48.487

49.347

50.312

51.322 51.641

52.382 52.998

54.554

55.577

56.967

58.798 59.232

60.312

61.265

62.710 63.323

64.101

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

[6]-

Sh

og

ao

l

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l

(-)-

Zin

gib

ere

ne

105

Appendix 21. HPLC of Acetone Extract Synthite Rajakumari Ginger

min

01

02

03

04

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\S

YN

TH

ITE

19

15

2.D

)

2.774 3.003 3.437 3.703

5.388

6.072 6.321 6.576

7.245 7.519 7.691 8.041

8.269 8.596

9.101 9.356 9.803 10.013 10.316 10.512

10.814

11.354 11.775 12.236

12.542 13.043 13.266

13.711 14.072

14.396 14.720 14.981 15.484 15.711 15.850 16.282 16.535 16.923

17.166 17.516

17.958 18.255

18.649

19.398 19.772

20.417 20.635 20.912 21.151 21.341 21.623 22.053

22.495 22.958

23.616 24.064 24.342

24.904 25.306

25.668 26.143

26.734 27.266

27.863 28.364

29.096 29.371

29.789 30.251

30.651 31.004

31.314

32.023

32.832 33.063

33.398 33.597 33.893

34.276 34.537

35.218

35.823

36.413

36.995

37.544 38.078

38.730 39.257

39.601

40.164 40.452 40.932

41.176 41.404 41.888 42.290

42.657

43.299 43.659

44.401

45.482 45.913

46.717 47.128

47.696

48.689 49.012

49.730

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l

[10

]-S

ho

ga

ol

[8]-

Sh

og

ao

l

[6]-

Sh

og

ao

l

106

Appendix 22. HPLC of Acetone Extract Synthite Irutti Ginger

min

05

10

15

20

25

30

35

40

45

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\S

YN

TH

ITE

19

15

3.D

)

2.163

2.734 2.997 3.431 3.777

5.213 5.249 5.397

6.074 6.331 6.588

7.255 7.528 7.687 8.038 8.263

8.595 9.105 9.360 9.532 9.825 10.016

10.321 10.524 10.822

11.265 11.781 12.245

12.547 13.049 13.264

13.707 14.063

14.382 14.708 14.993 15.494 15.855 16.301 16.539 16.925

17.178 17.521

17.955 18.261

18.651

19.388 19.761

20.389 20.903 21.144 21.435

22.035 22.466

22.947

23.672 24.110 24.329 24.714 24.876 25.283

25.647 26.117

27.244

27.838 28.345

29.083 29.351

29.768 30.231

30.627 30.988

31.298

32.009

32.814 33.049

33.388 33.578 33.751 34.264

34.521

35.196

35.805 36.085

36.394

36.975

37.523

38.074

38.714 39.245

39.592

40.158 40.447 40.912

41.175 41.394 41.884

42.284 42.652

43.291 43.639

44.394

45.462 45.896

46.691 47.099

47.663

48.657 48.979

49.692

[6]-

Sh

og

ao

l

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

[10

]-G

ing

ero

l

[8]-

Gin

ge

rol

[6]-

Gin

ge

rol

(-)-

Zin

gib

ere

ne

107

Appendix 23a. HPLC of Acetone Extract Synthite Nigerian Ginger

min

05

10

15

20

25

30

35

40

45

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\S

YN

TH

ITE

19

15

4.D

)

2.146

2.721 2.992 3.449

3.721 4.047

5.175 5.411

6.091 6.319

6.589

7.259 7.524 7.692

8.248 8.588

9.110 9.359 9.524 9.693 9.837 10.003

10.306 10.510 10.801

11.046 11.246 11.758 11.984 12.221

12.518 13.033 13.239 13.350

13.696 14.045

14.367 14.689 14.991 15.369 15.851 16.292

16.524 16.909

17.170 17.599

17.944 18.255

18.649 19.068

19.397 19.760

20.352

20.907 21.143 21.340 21.632 22.065 22.461

22.942

23.736 23.955 24.378 24.697 24.891

25.298 25.653

26.118

26.954 27.240

27.836 28.353

29.091 29.354

29.770 30.239

30.636 31.003

31.299

32.011

32.809 33.052

33.388 33.574

34.262 34.516

35.025 35.197 35.598 35.798

36.389

36.972

37.520

38.127

38.712 39.250

39.589

40.155 40.446 40.982 41.151

41.404 41.883 42.282

42.654

43.289

44.379

45.453 45.875

46.679 47.037

47.641

48.621 48.949

49.651

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l[6

]-S

ho

ga

ol

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

108

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

109

Appendix 24. HPLC of Acetone Extract Synthite Shimoga Ginger

min

05

1015

2025

3035

4045

mA

U 0

500

1000

1500

2000

2500

DA

D1

C, S

ig=2

80,1

6 R

ef=o

ff (G

ING

ER

\110

415S

L\S

YN

THIT

E19

155.

D)

2.144

2.752 2.839 3.007 3.260 3.463 3.748

5.185 5.384

6.317

7.163 7.515 8.013 8.265

8.588 9.105 9.366

9.870 10.027 10.526

10.813 11.255 11.406 11.789 12.222

12.535 13.041

13.273 13.681

14.052 14.203 14.395 14.668 14.905 15.366 15.539 15.753

16.332 16.631 16.905

17.169 17.506 17.599

17.939 18.258

19.389 19.764

20.340

20.902 21.184

21.453

22.079 22.567

22.946

23.706 24.178 24.720

25.017 25.288

25.653 26.122

27.241

27.877 28.335

28.902 29.095 29.341

29.751 30.245

30.642

31.283

31.995

32.777 33.055

33.602 33.884

34.238 34.502

34.964

35.788

36.390

36.940 37.235

37.484

38.049 38.314

38.679

39.221 39.555

40.117 40.402 40.553 40.706

40.953 41.350

41.830 42.226

42.587

43.206

44.301

45.347 45.766

46.545 46.966

47.505

48.475 48.786

49.534

[10]

-Sho

gaol

[8]-

Sho

gaol

[6]-

Sho

gaol

[6]-

Gin

gero

l

[8]-

Gin

gero

l

[10]

-Gin

gero

l

(-)-

Zin

gibe

rene

110

Appendix 25. HPLC of Acetone Extract Whole Herb Nigerian Ginger

min

05

10

15

20

25

30

35

40

45

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\W

HO

LE

HE

RB

-NIG

ER

IA.D

)

2.153

2.726 2.995 3.483 3.832

5.167 5.393

6.075 6.302 6.571

6.802 7.285

7.500

8.064 8.198 8.555 8.759

9.146 9.356 9.677 9.958 10.502

10.791 11.015 11.256 11.766 11.962 12.245

12.522 13.027 13.249 13.371

13.681 14.034

14.329 14.672 14.983 15.381 15.595 15.839

16.506 16.888

17.179 17.546

17.914 18.256

18.628 19.135

19.380 19.735

20.307

20.880 21.302 21.629 22.035 22.414 22.540

22.912

23.710 23.917 24.303 24.678 24.855

25.246 25.616

26.071

26.723 27.194

27.784 28.010

28.307

29.038 29.288

29.702 30.168

30.563 30.927

31.225

31.937

32.709 32.977

33.294 33.689

34.182 34.438

34.925 35.099

35.716

36.300

36.879 37.415

38.046 38.295

38.611 39.137

39.490

40.047 40.333

40.901 41.282 41.764 42.148

42.511

43.133

44.228

44.944 45.245 45.677 45.833

46.438 46.897

47.374

48.338 48.647

49.315

[8]-

Gin

ge

rol

[6]-

Gin

ge

rol

[10

]-G

ing

ero

l[6

]-S

ho

ga

ol

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

(-)-

Zin

gib

ere

ne

111

Appendix 26. HPLC of Acetone Extract Whole Herb Indian Ginger

min

05

10

15

20

25

30

35

40

45

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\W

HO

LE

HE

RB

-IN

DIA

.D)

2.738 3.008 3.388 3.490 3.861

5.152 5.409

6.055 6.300

6.556 6.783 7.228 7.499 7.996 8.218

8.563 8.754

9.358 9.535 9.674 9.987

10.273 10.502 10.790

11.253 11.435 11.755 12.245

12.526 13.030 13.242 13.506 13.688

14.040 14.336 14.672 15.016 15.405 15.590 15.845

16.396 16.519 16.912

17.191 17.542

17.933 18.272

18.647 19.185 19.396

19.771

20.411 20.907 21.127 21.325 21.651

22.023 22.435

22.940

23.717

24.319 24.687 24.875 25.036

25.272 25.636

26.096

27.217

27.797 28.045

28.323

29.056 29.309

29.720 30.183

30.577 30.952

31.254

31.962 32.228

32.749

33.323 33.707

34.208 34.448

35.124 35.510 35.731

36.021 36.325

36.896 37.433

38.023

38.627 39.155

39.505

40.069 40.356

40.925 41.326

41.790 41.928 42.173

42.532

43.149 43.517

44.275

45.400

46.472 46.946

47.440

48.396 48.730

49.456

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l

[10

]-S

ho

ga

ol

[8]-

Sh

og

ao

l

[6]-

Sh

og

ao

l

112

Appendix 27. HPLC of Acetone Extract Buderim Australian Ginger

min

05

10

15

20

25

30

35

40

45

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\B

UD

ER

IM A

US

TR

AL

IAN

.D)

2.179

2.729 2.995 3.457 3.787

5.347

6.059 6.282

6.540 6.825 7.233

7.480 7.980 8.197

8.546 8.743 9.070

9.343 9.512 9.851 10.012

10.242 10.491 10.773

11.243 11.540 11.749 12.223

12.510

13.073 13.240 13.490 13.689

14.034 14.326

14.657 14.957 15.370 15.601

15.836

16.523 16.916

17.193 17.555

17.938 18.268

18.674 19.064

19.415 19.773

20.317 20.598

20.909 21.318 21.651 22.050

22.556 22.902

23.702 23.925 24.343

24.854 25.242

25.635 26.095

26.638

27.195

27.850 28.033 28.325

29.061 29.293

29.720 30.194

30.582 30.751 31.250

31.763 32.118

32.423 32.725

32.997

33.548

34.205 34.462

34.932 35.172

35.730 35.966

36.318

36.900 37.432

37.857 38.059

38.625 39.153

39.501 39.862 40.057

40.343 40.659

40.910 41.272

41.773 42.175

42.538

43.157

43.808 44.242

44.616

45.287 45.699

46.467 46.902

47.427

48.367 48.653 49.093

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l

[6]-

Sh

og

ao

l

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

(-)-

Zin

gib

ere

ne

113

Appendix 28. HPLC of Acetone Extract Buderim Fijian Ginger

min

01

02

03

04

0

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\B

UD

ER

IM F

IJIA

N.D

)

2.144

2.729 2.997 3.498 3.778

5.225 5.370

6.085 6.312 6.576 6.837 7.263

7.497 8.000 8.199

8.559 8.751 9.105

9.352 9.514 9.854

10.249 10.493 10.774

11.234 11.753 12.220

12.510 13.037 13.231

13.669 14.029

14.329 14.651 14.940

15.356 15.829

16.291 16.509

16.902 17.187

17.550 17.928

18.258 18.642

19.062 19.402

19.754

20.310 20.614

20.897 21.314

21.638 22.062 22.564

22.902

23.930 24.326

24.852 25.243

25.631 26.083

26.394 26.633

27.179

28.037 28.319

29.054

29.717 30.199

30.584 30.950

31.246 31.752

32.133 32.424

32.724 32.996

33.285 33.549

34.199 34.468

34.930 35.163

35.722 35.959

36.305

36.888 37.420

38.159 38.301 38.621

39.234 39.496

39.851 40.054 40.345 40.669

40.921 41.275

41.774 42.176

42.538

43.156

43.847 44.241

44.993 45.274 45.714

46.472

47.417

48.378 48.662

49.353

(-)-

Zin

gib

ere

ne

[6]-

Gin

ge

rol

[8]-

Gin

ge

rol

[10

]-G

ing

ero

l[1

0]-

Sh

og

ao

l

[8]-

Sh

og

ao

l

[6]-

Sh

og

ao

l

114

Appendix 29. HPLC of Acetone Extract PharmaChem Chinese Ginger

min

05

10

15

20

25

30

35

40

45

mA

U 0

50

0

10

00

15

00

20

00

25

00

DA

D1

C,

Sig

=2

80

,16

Re

f=o

ff (

GIN

GE

R\1

10

41

5S

L\P

HA

RM

A C

HE

M.D

)

2.148

2.726 2.995 3.358 3.872

5.229 5.390

6.302 6.556 6.798 7.268 7.502 7.647 8.012 8.204

8.555 8.723 9.129 9.345

9.677 9.844 10.259

10.502 10.788

11.045 11.256 11.764 11.975 12.264

12.529

13.084 13.251 13.378 13.695 14.049

14.354 14.689 14.962 15.375 15.560 15.850

16.526 16.912

17.186 17.597

17.935 18.257

18.650

19.200 19.391 19.749

20.317

20.893 21.317

21.617 22.047 22.447

22.918

23.718 23.933 24.329

24.686 24.871 25.272

25.638 26.100

26.674

27.217

27.837 28.329

29.063 29.315

29.731 30.201

30.577 30.959

31.258

31.970

32.754 33.009

33.335 33.745

34.217 34.468

35.142 35.529

35.747 36.025

36.332

36.914 37.456

38.067

38.652 39.187

39.528

40.087 40.380

40.932 41.079 41.311 41.807

42.202 42.558

43.189 43.547 43.765

44.299

45.337 45.733

46.535 46.991

47.486

48.452 48.769

49.464

[6]-

Sh

og

ao

l

[8]-

Sh

og

ao

l

[10

]-S

ho

ga

ol

[10

]-G

ing

ero

l

[8]-

Gin

ge

rol

[6]-

Gin

ge

rol

(-)-

Zin

gib

ere

ne

115

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

Peak Area

Synthite

Rajakumari

Synthite

Irutti

Synthite

Nigerian

Synthite

Shimoga

Whole

Herb

Nigerian

Whole

Herb

Indian

Buderim

Australian

Buderim

Fijian

Pharma

Chem

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

0

100000

200000

300000

400000

500000

600000

0 4 8 11 15 18 22 25 29 33 37 40 44 48 53 56 61 65

Flu

ore

scen

ce

Time (minutes)

ORAC Fluorescence Decay Curve for Hexanes Extractions

BudAustralia

BudarimFijian

WH India

WHNigeria

PharmaChem

Synth Raj52

SynthIrutti 53

SynthNigeria 54

SynthShimoga55

116

Appendix 32. Acetone Fluorescence Decay Curve

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

0 4 8 11 15 18 22 25 29 33 37 40 44 48 53 56 61 65

Flu

ore

scen

ce

Time (minutes)

ORAC Fluorescence Decay Curve for Acetone Extractions

BudAustr

Bud Fij

WHIndia

WHNigeria

PCChineseSynthRaj 52

SynthIrutti 53

SynthNig 54

SynthShim55

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

14 16 18 20 22 24 26 28 30 32

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

19.56357.12708

18.85387.06351

20.59249.07585

18.83387.03937

17.02389.13327

19.29373.06696

19.20233.11540

17.98341.11169

19.92203.10791

25.72431.07260

26.14277.1043425.40

431.05734

33.13354.28436

32.90167.01237

30.34167.01225

27.78167.01230

16.61163.13234

24.11167.01225

15.20167.01230

19.71167.01230

NL: 5.10E6

Base Peak F: ITMS + c APCI corona Full ms [50.00-1000.00] MS F126-05%-5ul-newMeth

NL: 4.15E6

Base Peak F: ITMS + c APCI corona Full ms [50.00-1000.00] MS ginger-top5-f111-05%-10ul-newmethod

NL: 1.75E6

Base Peak F: ITMS + c APCI corona Full ms [50.00-1000.00] MS f142-05%-10ul-newmethod

NL: 4.11E6

Base Peak F: ITMS + c APCI corona Full ms [50.00-1000.00] MS f167-05%-5ul-newmeth

NL: 1.48E7

Base Peak F: FTMS + c ESI Full ms [100.00-1000.00] MS 500767005-f313

*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

14 16 18 20 22 24 26 28 30

Time (min)

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

18.01373.16418

18.26343.15396

17.19391.17484

18.99275.09116

14.43371.14859

30.56140.08153

27.57140.08156

26.43140.08156

24.35140.08159

22.30181.08566

18.87377.19553

19.17377.19537

18.51407.20575

20.56355.15375

18.15389.15909 21.55

193.0855328.92

140.0815627.60

140.0815125.91

140.0815615.01

371.14835

27.33277.17953

26.04297.20578

25.05261.18466

17.96334.23703

27.87275.16394

23.37140.08151

21.58115.08626

15.80117.06548

NL: 1.69E9

Base Peak F: FTMS + c APCI corona Full ms [100.00-1000.00] MS 500477605-108

NL: 1.45E9

Base Peak F: FTMS + c APCI corona Full ms [100.00-1000.00] MS 500477606-110

NL: 1.74E9

Base Peak F: FTMS + c APCI corona Full ms [100.00-1000.00] MS 500477607-165

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

15 16 17 18 19 20 21 22 23

Time (min)

20

40

60

80

100

Re

lative

Ab

un

da

nce

20

40

60

80

100

Re

lative

Ab

un

da

nce

19.57

357.21

18.91

387.22

20.61

249.2217.70

373.2418.33

179.1717.35

511.17

20.94

163.15

21.57

208.17

21.90

163.12

14.82

385.26

16.35

419.20

22.61

163.16

15.12

355.23

19.57

357.27

19.24

327.2920.18

371.23

20.63

163.13

21.20

163.12

22.30

163.15

23.28

140.14

18.25

140.13

17.82

140.13

17.33

140.14

15.48

163.19

15.74

140.16

16.45

140.13

14.95

140.16

NL: 4.99E6

Base Peak F: ITMS

+ c APCI corona Full

ms [50.00-1000.00]

MS gigner-F126

NL: 4.97E6

Base Peak F: ITMS

+ c APCI corona Full

ms [50.00-1000.00]

MS

hexahydrocurcumin

180 200 220 240 260 280 300 320 340 360 380

m/z

1000000

2000000

3000000

4000000

5000000

20

40

60

80

100

Re

lative

Ab

un

da

nce

357.24

177.17339.32 374.29179.18 207.20 233.26 313.35297.32249.46 271.46

357.24

177.18339.29 374.36179.17 207.17 221.30 233.32 313.37249.25 271.30 301.16290.32

NL: 4.78E6

gigner-F126#2737

RT: 19.61 AV: 1 F:

ITMS + c APCI corona

Full ms

[50.00-1000.00]

NL: 5.53E6

hexahydrocurcumin#25

68 RT: 19.55 AV: 1 F:

ITMS + c APCI corona

Full ms

[50.00-1000.00]

F126

hexahydrocurcumin

F126

hexahydrocurcumin

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

24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5

Time (min)

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

25.78

431.24

26.07

277.2525.45

431.22

27.42

249.1926.51

401.2827.07

275.3023.67

547.21

24.80

261.22

25.08

371.20

24.38

163.15

24.21

355.21

26.10

277.25

25.03

177.1727.75

223.17

27.45

140.19

23.89

140.12

24.06

140.17

25.85

140.1627.28

140.15

25.56

140.16

26.75

165.20

24.62

140.18

NL: 3.49E6

Base Peak F: ITMS

+ c APCI corona Full

ms [50.00-1000.00]

MS ginger-f167-apci

NL: 6.03E5

Base Peak F: ITMS

+ c APCI corona Full

ms [50.00-1000.00]

MS 6-gingerol-1k-5ul

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

m/z

200000

400000

600000

800000

20

40

60

80

100

Re

lative

Ab

un

da

nce

277.27

177.19

179.17259.29 293.27137.11 163.25 207.14 307.31220.19 249.40123.29

277.25

177.17

179.18259.28137.16 295.16163.16 249.34127.24 220.19207.22 307.36

NL: 1.80E6

ginger-f167-apci#3565

RT: 26.09 AV: 1 F:

ITMS + c APCI corona

Full ms

[50.00-1000.00]

NL: 8.06E5

6-gingerol-1k-

5ul#3471 RT: 26.10

AV: 1 F: ITMS + c APCI

corona Full ms

[50.00-1000.00]

F167

6-gingerol

F167

6-gingerol

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

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)

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

19.33

233.27

18.43

233.24 19.89

233.2220.41

233.2818.15

233.34

20.70

233.23

17.60

233.35

21.26

233.2617.29

233.31

21.71

233.30

16.76

233.35

15.32

233.31

16.36

233.30

19.34

233.24

18.43

233.22

17.46

233.2220.09

233.23 22.48

287.28

21.11

140.15

20.72

140.17

21.78

140.1617.78

140.16

16.99

140.15

16.51

140.18

15.67

164.15

15.41

140.12

NL: 1.48E6

Base Peak m/z=

232.50-233.50 F: ITMS +

c APCI corona Full ms

[50.00-1000.00] MS

ginger-f142-5k-pos

NL: 4.94E5

Base Peak F: ITMS + c

APCI corona Full ms

[50.00-1000.00] MS

4-gingerdiol-1k-5ul

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

m/z

200000

400000

600000

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

233.22

269.07

163.12

166.28 237.24219.21137.15 251.24209.18 292.31191.22152.26 303.35125.16

233.24

269.09

163.15

251.17137.13177.23 292.26201.32 249.28151.16127.23 219.27 312.35263.29

NL: 9.79E5

ginger-f142-5k-

pos#2910 RT: 19.25

AV: 1 F: ITMS + c APCI

corona Full ms

[50.00-1000.00]

NL: 6.83E5

4-gingerdiol-1k-

5ul#2597 RT: 19.34

AV: 1 F: ITMS + c APCI

corona Full ms

[50.00-1000.00]

F142

4-gingerdiol

F142

4-gingerdiol

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

14 15 16 17 18 19 20 21 22 23

Time (min)

20

40

60

80

100

Rela

tive A

bundance

20

40

60

80

100

Rela

tive A

bundance

17.13

371.29 17.30

371.31

19.36

355.29

17.99

377.11

16.89

373.2418.31

355.2815.39

447.1619.58

371.2115.74

507.1320.39

193.1621.06

181.13

13.85

371.2521.43

371.27

14.22

563.2322.28

163.20

22.74

163.15

17.73

341.29

17.97

341.2317.36

341.2818.38

341.2719.15

341.25

19.72

341.33

20.36

341.31

21.20

341.28

22.52

341.29

22.19

341.33

16.59

341.29

16.09

341.34

15.59

341.31

14.64

341.34

14.00

341.40

NL: 1.17E6

Base Peak F: ITMS + c

APCI corona Full ms

[50.00-1000.00] MS

ginger-f110-apci

NL: 1.23E5

Base Peak m/z=

340.50-341.50 F: ITMS

+ c APCI corona Full

ms [50.00-1000.00]

MS ohc-100

100 150 200 250 300 350 400 450 500 550 600 650

m/z

0

20000

40000

60000

80000

100000

120000

0

20

40

60

80

100

Rela

tive A

bundance

341.30

377.02

163.16217.23137.13 477.33 513.07400.29 431.31309.37 537.36285.29 583.30 635.12

341.28

377.04

163.15137.12 217.24400.30 431.31309.21 477.28 513.04131.19 279.36 543.50 615.36 651.27

NL: 9.57E5

ginger-f110-apci#2246

RT: 17.72 AV: 1 F:

ITMS + c APCI corona

Full ms [50.00-1000.00]

NL: 1.22E5

ohc-100#2126 RT:

17.71 AV: 1 F: ITMS +

c APCI corona Full ms

[50.00-1000.00]

F110

octahydrocurcumin

F110

octahydrocurcumin

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

21 22 23 24 25 26 27 28 29

Time (min)

0

20

40

60

80

100

Rela

tive A

bundance

0

20

40

60

80

100

Rela

tive A

bundance

26.12

277.23

24.79

261.30

25.79

295.2623.79

387.2121.78

533.2326.68

275.23

23.11

431.1927.86

140.17

21.29

533.1828.69

140.15

22.82

140.1721.02

140.16

24.80

261.2923.80

261.26

25.26

261.2322.78

261.2520.46

137.1128.89

140.16

27.59

140.15

26.88

140.16

27.91

140.15

26.50

140.1324.54

140.1522.97

140.13

22.06

140.12

21.51

140.16

NL: 3.82E6

m/z= 53.91-624.18 F:

ITMS + c APCI corona

Full ms [50.00-1000.00]

MS ginger-f165-5k-pos

NL: 8.62E4

Base Peak F: ITMS + c

APCI corona Full ms

[50.00-1000.00] MS

6-gingerdiol-100

100 150 200 250 300 350 400

m/z

0

20000

40000

60000

0

20

40

60

80

100

Rela

tive A

bundance

261.30

297.11

279.20163.20

137.14 320.35177.15 351.34 397.34229.36 377.31191.21 415.35

261.30

297.09

279.19

163.19137.15

177.23 320.34229.35 391.18351.39191.29 411.44113.18

NL: 6.36E5

ginger-f165-5k-pos#3031

RT: 24.79 AV: 1 F:

ITMS + c APCI corona

Full ms [50.00-1000.00]

NL: 7.83E4

6-gingerdiol-100#2998

RT: 24.82 AV: 1 F:

ITMS + c APCI corona

Full ms [50.00-1000.00]

F16

5

6-

gingerdiol

F16

5

6-

gingerdiol

128

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

129

Appendix 46. Optimizing Antioxidant Concentrations Using ORAC Assay

Testing

Levels:4-Gingerol

0.001%

4-Gingerol

0.002%

4-Gingerol

0.004%

4-Gingerol

0.006%

4-Gingerol

0.008%

4-Gingerol

0.01%

4-Gingerol

0.02%

4-Gingerol

0.04%

4-Gingerol

0.06%

4-Gingerol

0.08%

4-Gingerol

0.1%

4-Gingerol

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

Levels:4-Shogaol

0.001%

4-Shogaol

0.002%

4-Shogaol

0.004%

4-Shogaol

0.006%

4-Shogaol

0.008%

4-Shogaol

0.01%

4-Shogaol

0.02%

4-Shogaol

0.04%

4-Shogaol

0.06%

4-Shogaol

0.08%

4-Shogaol

0.1%

4-Shogaol

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

Testing

Levels:

4-

Gingerdiol

0.001%

4-

Gingerdiol

0.002%

4-

Gingerdiol

0.004%

4-

Gingerdiol

0.006%

4-

Gingerdiol

0.008%

4-

Gingerdiol

0.01%

4-

Gingerdiol

0.02%

4-

Gingerdiol

0.04%

4-

Gingerdiol

0.06%

4-

Gingerdiol

0.08%

4-

Gingerdiol

0.1%

4-

Gingerdiol

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

Levels:6-Gingerol

0.006%

6-Gingerol

0.008%

6-Gingerol

0.01%

6-Gingerol

0.02%

6-Gingerol

0.04%

6-Gingerol

0.06%

6-Gingerol

0.08%

6-Gingerol

0.1%

6-Gingerol

0.2%

6-Gingerol

0.3%

6-Gingerol

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

Testing

Levels:

S-6-

Gingerol

0.008%

S-6-

Gingerol

0.02%

S-6-

Gingerol

0.1%

S-6-

Gingerol

0.6%

Net

AUC: 16.4926 18.5079 23.6127 18.5701

Testing

Levels:

6-

Gingerdiol

0.006%

6-

Gingerdiol

0.008%

6-

Gingerdiol

0.01%

6-

Gingerdiol

0.02%

6-

Gingerdiol

0.04%

6-

Gingerdiol

0.06%

6-

Gingerdiol

0.08%

6-

Gingerdiol

0.1%

6-

Gingerdiol

0.2%

6-

Gingerdiol

0.3%

6-

Gingerdiol

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

Levels:THC

0.0001%

THC

0.001%

THC

0.002%

THC

0.004%

THC

0.006%

THC

0.008%

THC

0.01%

THC

0.02%

THC

0.04%

THC

0.06%

THC

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

Levels:THC

0.1%

THC

0.3%

THC

0.5%

THC

0.8%

THC

1%

HHC

0.0001%

HHC

0.001%

HHC

0.01%

HHC

0.1%

HHC

1%

Net

AUC: 15.2247 14.75 14.7461 14.634 16.0468 -3.8236 2.21764 15.3073 15.5896 17.9958

Testing

Levels:OHC

0.0001%

OHC

0.001%

OHC

0.002%

OHC

0.004%

OHC

0.006%

OHC

0.008%

OHC

0.01%

OHC

0.02%

OHC

0.04%

OHC

0.06%

OHC

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

Levels:OHC

0.1%

OHC

0.3%

OHC

0.5%

OHC

0.8%

OHC

1%

Net

AUC: 15.499 15.964 15.6885 16.3023 17.7887

Testing

Levels:

Toco-

pherols

0.00006%

Toco-

pherols

0.0001%

Toco-

pherols

0.0005%

Toco-

pherols

0.001%

Toco-

pherols

0.003%

Toco-

pherols

0.005%

Toco-

pherols

0.008%

Toco-

pherols

0.01%

Toco-

pherols

0.03%

Toco-

pherols

0.05%

Toco-

pherols

0.08%

Toco-

pherols

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

130

Appendix 47. LCMS of Tetrahydrocurcumin

131

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

132

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

133

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

Anti-

oxidant

Dilution

Used

Amt. of

Dilution

(g)

Filled

to (g)

Target

Conc.

Actual

Conc.

(ppm)

Amt. of Oil

(g) for PV

Titrant

Volume

(mL)

Peroxide

Value

Aver-

age P.V.

Antioxid

ant Conc.

in Bvg

4-

gingerol,

A

0.1% in

EtOH

0.108 7.996 12.5 ppm 13.51 5.02 10.55 20.32 21.84

4-

gingerol,

B

0.1% in

EtOH

0.099 7.996 12.5 ppm 12.38 5.00 12.57 24.44

4-

gingero

l, C

0.1% in

EtOH

0.102 7.997 12.5 ppm 12.75 5.01 10.75 20.76 1.25ppb

4-

gingerdi

ol, A

0.1% in

EtOH

0.1 7.994 12.5 ppm 12.51 5.02 12.35 23.90 22.87

4-

gingerd

iol, B

0.1% in

EtOH

0.1 7.998 12.5 ppm 12.50 5.01 11.75 22.75 1.25ppb

4-

gingerdi

ol, C

0.1% in

EtOH

0.106 7.998 12.5 ppm 13.25 5.04 11.42 21.96

4-

shogaol,

A

0.1% in

EtOH

0.108 7.996 12.5 ppm 13.51 5.03 10.82 20.82 21.72

4-

shogaol,

B

0.1% in

EtOH

0.1 8.087 12.5 ppm 12.37 5.01 11.62 22.50

4-

shogaol,

C

0.1% in

EtOH

0.107 8.002 12.5 ppm 13.37 5.01 11.29 21.84 1.25ppb

6-

gingerol,

A

1% in

EtOH

0.104 8.02 125 ppm 129.68 5.01 12.81 24.87 23.04

6-

gingero

l, B

1% in

EtOH

0.101 8.008 125 ppm 126.12 5.00 11.56 22.42 12.5ppb

6-

gingerol,

C

1% in

EtOH

0.108 7.997 125 ppm 135.05 5.00 11.26 21.82

S-6-

gingerol,

A

0.1% in

Acetone

1.002 8.177 125 ppm 122.54 5.18 17.64 33.38 34.79

S-6-

gingerol,

B

0.1% in

Acetone

0.989 8.304 125 ppm 119.10 5.00 19.42 38.14

S-6-

gingerol,

C

0.1% in

Acetone

1.007 8.129 125 ppm 123.88 5.01 16.81 32.85

6-

gingerd

iol, A

1% in

EtOH

0.064 8.03 75 ppm 79.70 5.01 11.90 23.05 21.40 12.5ppb

6-

gingerdi

ol, B

1% in

EtOH

0.06 8.008 75 ppm 74.93 5.00 12.00 23.30

134

6-

gingerdi

ol, C

1% in

EtOH

0.065 7.995 75 ppm 81.30 5.00 9.28 17.86

6-

shogaol,

A

1% in

EtOH

0.099 8.017 125 ppm 123.49 5.00 13.01 25.32 25.65 12.5ppb

6-

shogaol,

B

1% in

EtOH

0.101 8.008 125 ppm 126.12 5.07 14.64 28.19

6-

shogaol,

C

1% in

EtOH

0.115 8.02 125 ppm 143.39 5.01 12.10 23.45

8-

gingerol,

A

1% in

EtOH

0.1 8.002 125 ppm 124.97 5.01 14.98 29.20 25.32

8-

gingero

l, B

1% in

EtOH

0.097 8.021 125 ppm 120.93 5.07 12.44 23.85 12.5ppb

8-

gingerol,

C

1% in

EtOH

0.101 8.015 125 ppm 126.01 5.01 11.83 22.91

8-

shogaol,

A

1% in

EtOH

0.099 8.004 125 ppm 123.69 5.03 10.82 20.82 21.03 12.5ppb

8-

shogaol,

B

1% in

EtOH

0.099 8.01 125 ppm 123.60 5.02 10.35 19.92

8-

shogaol,

C

1% in

EtOH

0.104 8.009 125 ppm 129.85 5.02 11.57 22.35

10-

gingerol,

A

1% in

EtOH

0.099 8.063 125 ppm 122.78 5.01 11.88 23.01 24.18

10-

gingerol,

B

1% in

EtOH

0.105 7.996 125 ppm 131.32 5.01 13.12 25.49

10-

gingero

l, C

1% in

EtOH

0.098 8.006 125 ppm 122.41 5.03 12.44 24.04 12.5ppb

10-

shogaol,

A

1% in

EtOH

0.102 8.003 125 ppm 127.45 5.08 15.12 29.07 26.69

10-

shogaol,

B

1% in

EtOH

0.1 7.998 125 ppm 125.03 5.07 13.60 26.13 12.5ppb

10-

shogaol,

C

1% in

EtOH

0.103 7.992 125 ppm 128.88 5.02 12.83 24.86

Tetrahy

drocurc

umin, A

1% in

EtOH

0.044 7.992 50 ppm 55.06 5.00 9.13 17.56 18.68

Tetrahy

drocurc

umin, B

1% in

EtOH

0.041 8 50 ppm 51.25 5.01 9.29 17.84 5ppb

Tetrahy

drocurc

umin, C

1% in

EtOH

0.046 8.006 50 ppm 57.46 5.14 10.95 20.62

135

Hexahyd

rocurcu

min, A

1% in

Acetone

0.102 8.005 125 ppm 127.42 5.02 12.51 24.22 21.49

Hexahy

drocurc

umin, B

1% in

Acetone

0.1 8.002 125 ppm 124.97 5.00 10.42 20.14 12.5ppb

Hexahyd

rocurcu

min, C

1% in

Acetone

0.101 8.009 125 ppm 126.11 5.01 10.42 20.10

Octahyd

rocurcu

min, A

0.1% in

EtOH

0.214 8.004 25 ppm 26.74 5.08 15.03 28.90 29.49 2.5ppb

Octahyd

rocurcu

min, B

0.1% in

EtOH

0.204 7.994 25 ppm 25.52 5.04 14.25 27.58

Octahyd

rocurcu

min, C

0.1% in

EtOH

0.203 8.02 25 ppm 25.31 5.03 16.44 31.99

50%

Tocoph

erols, A

10% in

EtOH

0.04 8.044 500 ppm 497.27 5.00 6.81 12.92 15.26 25ppb

50%

Tocophe

rols, B

10% in

EtOH

0.048 8.105 500 ppm 592.23 5.00 10.62 20.54

50%

Tocophe

rols, C

10% in

EtOH

0.041 8.015 500 ppm 511.54 5.02 6.53 12.31

Blank,

Refriger

ated, A

8.07 5.03 7.07 13.35 Old

Pipette

Blank,

Refriger

ated, B

8.348 5.00 7.46 14.21 Old

Pipette

Blank,

Refriger

ated, C

8.084 5.04 7.12 13.43 Old

Pipette

Blank,

Refriger

ated, D

(New

pipette)

5.10 8.44 15.86 15.86 0

Blank,

Hot Box,

A

8.136 5.00 6.50 12.30 Old

Pipette

Blank,

Hot

Box, B

8.004 5.00 11.19 21.68 19.10 0

Blank,

Hot Box,

C

8.089 5.02 8.64 16.51

*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.

136

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

Anti-

oxidant

Dilution

Used

Amt. of

Dilution

(g)

Filled

to (g)

Target

Conc.

Actual

Conc.

(ppm)

Amt. of Oil

(g) for PV

Titrant

Volume

(mL)

Peroxide

Value

Aver-age

P.V.

4-gingerol, A

1% in EtOH

0.078 7.995 0.01% 97.56 5.00 9.20 18.00 20.20

4-gingerol, B

1% in EtOH

0.079 8.064 0.01% 97.97 5.01 11.90 23.35

4-gingerol, C

1% in EtOH

0.083 8.005 0.01% 103.69 4.99 9.80 19.24

4-gingerdiol,

A

1% in EtOH

0.077 7.999 0.01% 96.26 5.00 11.60 22.80 24.59

4-gingerdiol,

B

1% in EtOH

0.081 8.02 0.01% 101.00 5.00 12.20 24.00

4-gingerdiol,

C

1% in EtOH

0.088 8.111 0.01% 108.49 5.00 13.69 26.98

4-shogaol, A

1% in EtOH

0.08 8.001 0.01% 99.99 4.99 11.73 23.11 22.66

4-shogaol, B

1% in EtOH

0.081 7.989 0.01% 101.39 5.00 11.27 22.14

4-shogaol, C

1% in EtOH

0.082 8.092 0.01% 101.33 5.00 11.56 22.72

6-gingerol, A

1% in EtOH

0.798 8.066 0.10% 989.34 5.01 20.19 39.90 38.78

6-gingerol, B

1% in EtOH

0.829 8.214 0.10% 1009.25 5.01 22.17 43.85

6-gingerol, C

1% in EtOH

0.853 8.12 0.10% 1050.49 5.11 16.85 32.58

6-gingerdiol,

A

1% in EtOH

0.48 8.008 0.06% 599.40 5.00 20.00 39.60 35.48

6-gingerdiol,

B

1% in EtOH

0.48 8.05 0.06% 596.27 5.00 17.42 34.44

6-gingerdiol,

C

1% in EtOH

0.477 8.151 0.06% 585.20 4.99 16.37 32.40

Tetrahydrocurcumin,

A

1% in EtOH

0.317 7.997 0.04% 396.40 5.01 17.03 33.59 30.80

Tetrahydrocurcumin,

B

1% in EtOH

0.326 8.026 0.04% 406.18 5.01 14.84 29.22

Tetrahydrocurcumin,

C

1% in EtOH

0.322 7.997 0.04% 402.65 5.00 15.00 29.60

Octahydrocurcumin, A

1% in EtOH

0.158 7.996 0.02% 197.60 5.01 11.15 21.86

Octahydrocurcumin, B

1% in EtOH

0.16 8.008 0.02% 199.80 5.00 14.52 28.64 28.53

Octahydrocurcumin, C

1% in EtOH

0.166 8.151 0.02% 203.66 5.06 14.58 28.42

Tocopherols, A

10% in EtOH

0.158 7.995 0.10% 1976.24 5.00 13.47 26.54 25.96

Tocopherols, B

10% in EtOH

0.161 8 0.10% 2012.50 4.99 12.18 24.01

Tocopherols, C

10% in EtOH

0.16 7.991 0.10% 2002.25 5.01 13.89 27.33

137

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

138

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

139

Appendix 54. Bar Chart to Display Significance of Paired Comparisons

140

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

Argentinian Lemon 1X

p-Cymene Limonene Limonene

oxide -Terpineol Geranial Neral Carvone

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

141

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

142

Appendix 56. GC of Original Starting Argentinian Lemon Oil