Sappanwood Water Extract

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Title:
Sappanwood Water Extract Evaluation of Color Properties, Functional Properties, and Toxicity
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1 online resource (108 p.)
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english
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Sinsawasdi, Valeeratana K
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University of Florida
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Simonne, Amarat H
Committee Members:
Gu, Liwei
Marshall, Maurice R
Wysocki, Allen F

Subjects

Subjects / Keywords:
ames-mutagenicity-test -- antimicrobial -- antioxidant -- caesalpinia-sappan-l -- chromaticity -- colorant -- food -- red -- sappanwood
Food Science and Human Nutrition -- Dissertations, Academic -- UF
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Food Science and Human Nutrition thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The red color from water extracts of sappanwood (heartwood of Caesalpinia sappan L.) has been used in Thai and Indonesian beverages for hundreds of years. The colorant may have potential as an alternative for synthetic red dyes. However, scientific data on this red colorant is limited. Water extracts of Caesalpenia sappan prepared from different methods (reflux at 5h (RF 5); 24h (RF 24); room temperature extract at 6 h (RT)) were evaluated for color, functional properties, and toxicity. Color properties were described by CIE L*a*b* measurements, hue angle, chroma, and UV-vis spectra. Total phenolic (TP) content and antioxidant activities were evaluated by the Folin-Ciocalteu colorimetric method, and by the oxygen radical absorbance capacity (ORAC) and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assays, respectively. Mutagenicity was determined by the Ames assay (with and without metabolic activation) while antimicrobial activity was determined by disc diffusion method. In contrast to other naturally derived red pigments such as anthocyanins, the color of C. sappan pigments is red at pH 8 - 12, and appears yellow at lower pH. Brazilein and brazilin, the compounds responsible for giving red color to the extract, were detected and identified by a high performance liquid chromatography and mass spectroscopy (LC-MS). Total phenolic content and antioxidant activities in RF 24 were not significantly higher than RF5, and thus RF24 was excluded from further evaluation. The extracts (RF5 and RT) showed no mutagenicity. The extracts exhibited antimicrobial activity against common spoilage bacteria including Alcaligenes faecali, Bacillus coagulans, and Pseudomonas puida at a concentration of 50 mg/mL. The stability of color to heat, fluorescent light and storage temperature at different pH levels was examined. The hue of the extract was affected by concentration (0.005 - 0.4000 mg/mL) but the visual color remained in pink to red shade for pH 8 and 9. Results indicated that the color from sappanwood extract was more stable at higher pH (9) than at lower pH (7). Overall, results from this current study show the potential for developing sappanwood water extract into a naturally derived red color additive for non-acidic food products.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Valeeratana K Sinsawasdi.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Simonne, Amarat H.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-05-31

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1 SAPPANWOOD WATER EXTRACT: EVALUATION OF COLOR PROPERTIES, FUNCTIONAL PROPERTIES, AND TOXICITY By VALEERATANA KALANI SINSAWASDI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILL MENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Valeeratana Kalani Sinsawasdi

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3 To my mother Pimprapai Kunchavalee Sinsawasdi, and my father, Dr. Narong Sinsawasdi

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4 ACKNOWLEDGMENTS I w ould like to thank my thesis advisor Dr. Amarat Simonne, for the trust she had in me from the beginning and also for her guidance and partial financial support throughout this journey. I would like to thank my committee members, Dr. Maurice R. Marshall f or his patien ce in answering my questions and laboratory support; Dr. Liwei Gu for the use of his mass spectrometry lab; and Dr. Allen F. Wys ocki for inspiration I would like to thank Mahidol University International College for its partial financial supp ort, and also Dr. Maleeya Kruatrachue, Dr. Yaowalark Sukthana, and Dr. Nirutchara Laohaprasit who help ed and supp orted me beyond my expectation s S pecial thanks also go to Wei Yea Hsu for her great advi ce and support, which helped me and my daughter enjoy living in Gainesville for the past three years. Many thanks also go to Bridget L. Stokes, Milena Ramirez, Yavuz Ya giz Amandeep K. Sandhu, Jodie Johnson, and Paul A. Proctor for their technical support during the various stages of my research. I would li ke to thank all my friends at the Food Science and Human Nutrition Department, especially Lemanne Delva, and Jinlan Zhao for their wonderful friendship. I thank my husband, Tawarit Warit, for his understanding. My heartfelt thanks go to my daughter, Pw arita Nalani Warit who fills my heart everyday with happiness while I have finished my studies Finally I am grateful to have been born to my mother, Pimprapai Sinsawasdi, and my fa ther, Dr. Narong Sinsawasdi, because they have always provided me with ev erything I ever wanted and have demonstrated unconditional love and support. My most special thanks go to my mother who decided to discontinue her Ph.D. study at the University of Hawaii to make sure I received the best of everything she could provide fro m the start of my life.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF F IGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUC TION ................................ ................................ ................................ .... 14 Justification ................................ ................................ ................................ ............. 14 Hypothesis ................................ ................................ ................................ .............. 15 Specific Objectives ................................ ................................ ................................ 16 2 LITERATURE REVIEW ................................ ................................ .......................... 17 Caesalpinia Sappan ................................ ................................ ................................ 17 History of Consumptio n ................................ ................................ .................... 17 Color Properties of Sappanwood Extracts ................................ ........................ 19 Antioxidant Activities of Sappanwood Extracts ................................ ................. 20 Antimicrobial and Probiotic Activities of Sappanwood Extracts ........................ 24 Medicinal Benefits of Sappanwood Extracts ................................ ..................... 25 Chemical Compounds Isolated from C. sappan Heartwood ............................. 29 Sappanwood Extract as a Food Additive ................................ .......................... 31 Conclusion ................................ ................................ ................................ .............. 33 3 ANTIOXIDANT CAPACITIES AND COLOR CHARACTERISTICS OF SAPPANWOOD WATER EXTRACTS ................................ ................................ .... 38 Background Overview ................................ ................................ ............................. 38 Materials and Methods ................................ ................................ ............................ 40 Raw Materials and Chemicals ................................ ................................ .......... 40 Extracts Preparation ................................ ................................ ......................... 40 Size reduction of the heartwood chips ................................ ....................... 40 Water extraction of sappanwood ................................ ................................ 41 Folin Ciocalteu Assay for Total Phenolic Content ................................ ............ 42 Antioxidant Activity ................................ ................................ ........................... 42 DPPH Radical Scavenging Activity ................................ ............................ 42 Oxygen Radical Absorbance Capacity (ORAC) ................................ ......... 43 Chromaticity of Sappanwood Water Extract ................................ ..................... 43

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6 Brazilein Analysis (HPLC DAD ESI MS n Analysis) ................................ ........... 44 Statistical Analysis ................................ ................................ ............................ 44 Results and Discussion ................................ ................................ ........................... 45 Total Phenolic Content and Antioxidant Capacities ................................ .......... 45 Chromaticity of Sappanwood Water Extract ................................ ..................... 46 Conclusion ................................ ................................ ................................ .............. 48 4 AMES MUTAGENICITY ASSAY AND ANTIMICROBIAL ACTIVITY OF SAPPANWOOD WATER EXTRACTS ................................ ................................ .... 55 Background O verview ................................ ................................ ............................. 55 Materials and Methods ................................ ................................ ............................ 59 Sappanwood Extracts Preparation ................................ ................................ ... 59 Chemicals and Microbiological Media ................................ .............................. 59 Bacteria Strains ................................ ................................ ................................ 60 Ames Mutagenicity Assay ................................ ................................ ................ 60 Antimicrobial Activity by Standard Agar Disc Diffusion Method ........................ 62 Statistical Analysis ................................ ................................ ............................ 63 Results an d Discussion ................................ ................................ ........................... 63 Ames Mutagenicity Assay ................................ ................................ ................ 63 Antimicrobial Activity Evaluated by Disc Diffusion Method ............................... 65 Conclusion ................................ ................................ ................................ .............. 67 5 STABILITY OF RED COLOR FROM SAPPANWOOD COLD WATER EXTRACT IN AQUEOUS SOLUTIONS ................................ ................................ .. 72 Background Overview ................................ ................................ ............................. 72 Materials and Methods ................................ ................................ ............................ 73 Raw Materials and Chemicals ................................ ................................ .......... 73 Sappanwood Extracts Preparation ................................ ................................ ... 74 Color Measurements ................................ ................................ ........................ 74 Spectrophotometric Analysis ................................ ................................ ............ 75 Color Stability Study of Sappanwood Extracts Under Different Conditions ...... 75 Thermostability ................................ ................................ ........................... 75 Effects of concentrations on chromaticity ................................ ................... 76 Storage stability ................................ ................................ ......................... 76 Statistical Analysis ................................ ................................ ............................ 77 Results and Discussion ................................ ................................ ........................... 77 Thermostability ................................ ................................ ................................ 77 Concentration Effects on Color of Sappanwo od Extracts ................................ 78 Storage Stability ................................ ................................ ............................... 80 Conclusion ................................ ................................ ................................ .............. 81 6 SUMMARY AND CONCLUSIONS ................................ ................................ .......... 87

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7 APPENDIX A EFFECTS OF EXTRACTION TIME ON MAXIMUM ABSORBANCE OF SAPPANWOOD COLD WATER EXTRACTION ................................ ..................... 88 Background ................................ ................................ ................................ ............. 88 Materials and Methods ................................ ................................ ............................ 88 Results and Discussion ................................ ................................ ........................... 88 B EXAMPLE OF STATISTICAL ANALYSIS ................................ ............................... 90 C PREPARAT ION OF SAPPANWOOD EXTRACT SOLUTIONS AT VARIOUS CONCENTRATIONS FOR COLOR STABILITY TESTING ................................ ..... 91 D CHEMICAL OXIDATION OF BRAZILIN TO BRAZILEIN ................................ ........ 92 Background ................................ ................................ ................................ ............. 92 Materials and Methods ................................ ................................ ............................ 92 Results and Discussion ................................ ................................ ........................... 93 LIST OF REFERENCES ................................ ................................ ............................... 99 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 108

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8 LIST OF TABLES Table page 2 1 Summary of antioxidant activities and benefits of various C. sappan heartwood extracts in foods ................................ ................................ ................ 22 3 1 Buffer preparation for chromaticity testing ................................ .......................... 50 3 2 Total polyphenol content and antioxidant activities of sappanwood water extracts (RF5, RF24, RT) ................................ ................................ ................... 50 3 3 Chroma of sappanwood water extracts at pH 9. ................................ ................. 51 4 1 Mutagenic dose response of RF5 sappanwood water extract to Salmonella Typhimurium (TA98 and TA100 ) as represented by the mean number of revertant colonies (CFU/plate) +/ standard deviation (n=3) ............................... 69 4 2 Mutagenic dose response of RT sappanwood water extract to Salmonella Typhimurium (TA98 and TA100) as represented by the mean number of revertant colonies +/ standard deviation (n=3) ................................ .................. 70 4 3 Antibacterial activities of sappanwood water extracts against three strains of food spoi lage bacteria. ................................ ................................ ....................... 71 5 1 Effects of various concentrations of sappanwood water extracts on hue angle at pH 7, 8, and 9 ................................ ................................ ................................ 84 5 2 Color differences during storage of sappanwood extracts under various storage conditions and pH ................................ ................................ .................. 86 C 1 Preparation of sappanwood extract solutions at various concentrations ............ 91

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9 LIST OF FIGURES Figure page 2 1 Molecular structures of brazilin (left) and brazilein (right). ................................ .. 34 2 2 Pro posed mechanism of red shift of brazilein when 0.1N NaOH was added ...... 34 2 3 1',4' dihydro spiro[benzofuran 3(2H),3' [3H 2]benzopyran] 1',6',6',7' tetrol ........ 34 2 4 3 [[4,5 dihydroxy 2(hydroxymethyl) phenyl] methyl] 2,3 dihydro 3,6 benzofurandiol ................................ ................................ ................................ .... 35 2 5 Protosappanin A ................................ ................................ ................................ 35 2 6 Protosappanin B and protosappanin C ................................ ............................... 35 2 7 Protosappanin D ................................ ................................ ................................ 36 2 8 Protosappanin E ................................ ................................ ................................ 36 2 9 Sappanchalcone ................................ ................................ ................................ 36 2 10 7 hydroxy 3 hydroxybenzylidene) chroman 4 one ................................ ........ 37 2 11 Brazilid e A ................................ ................................ ................................ .......... 37 2 12 trihydroxy 3 benzyl 2 H chromene ................................ ............................ 37 3 1 Wiley Mill (Thomas Wiley Lab Mill Model 4). ................................ ...................... 49 3 2 Metal screen with 2 mm diameter attached to the Wiley Mill (Thomas Wiley Lab Mill Model 4). ................................ ................................ ............................... 49 3 3 Sappanwood powder ground from sappanwood chips. ................................ ...... 49 3 4 Positive ion electrospray product ion mass spectra of brazilein ........................ 51 3 5 Visual colors of aqueous solution of sappanwood extracts (RF5, RF24, RT) at pH 9. Photos from top to bottom are RF5, RF24, and RT. ............................ 52 3 6 Redness of sappanwood water extracts represented by mean a* value (n = 3). ................................ ................................ ................................ ....................... 52 3 7 Chromaticity as a*, b* and hue angles of SPWE. The hue angles of SPWE pH 2 5 were low in a* but high in b* (data points are in the brown circle).. ......... 5 3 3 8 UV vis spectra of sappanwood (RT) extract showing bathochromic shift at pH 7 and higher. ................................ ................................ ................................ ....... 54

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10 3 9 Absorbance of sappanwood extracts RT at 525 and 535 nm in buffer solutions pH 2 12. ................................ ................................ ............................ 54 4 1 5 hydroxy 1,4 naphthoquinone ................................ ................................ ........... 68 5 1 Effects of pH (2, 5, 7, 9, and 12) on heat stability of sappanwood extracts in terms o f a* value (redness) with light and air exposure ................................ ...... 82 5 2 UV vis spectra of sappanwood extract at pH 9 during heating (80 C) with light and air exposure. ................................ ................................ ........................ 82 5 3 Effects of heat on % color retention of sappanwood extracts (at 539 nm) at pH 9 under different environments. ................................ ................................ .... 83 5 4 Effects of sappanwood extract concentration s on color saturation (chroma) at pH 7, 8 and 9. ................................ ................................ ................................ ..... 83 5 5 Visual colors of different sappanwood extract concentrations at pH 7, 8, and 9 (high to low from left to right). ................................ ................................ .......... 84 5 6 Sappanwood water extract after 35 days in different environments, from left to right of each picture, at 25 C with light exposure, at 25 C in the dark, and at 4 C in the dark. ................................ ................................ .............................. 85 A 1 Effects of extraction time on UV vis spectra (from 400 to 600 nm) of sappanwood cold water extracts. ................................ ................................ ....... 89 A 2 Effects of extraction time on absorbance a t 445 nm of cold water extracts of sappanwood powder. Data represents the mean of n=3. Columns with .................. 89 D 1 Chromatog ram of brazilin sample analyzed via C18 HPLC/UV/( )ESI MS n Brazilin and brazilein ion peaks are shown in the shaded area .......................... 95 D 2 Chromatogram of oxidized brazilin sample via C18 HPLC/UV/( )ES I MS n There are 2 brazilein ion peaks which are shown in shaded area. ..................... 96 D 3 Mass spectrum of brazilin (top) sample and oxidized brazilin sample (bottom) with ( )ESI MS ................................ ................................ ................................ .... 97 D 4 The [M H] ions of brazilin (top) and brazilein (bottom) were disassociated to produce a number of product ions. ................................ ................................ ..... 98

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11 LIST OF ABBREVIATION S CFU Colony forming unit DPPH 2, 2 D iphenyl 1 picrylhydrazyl ET Electron transfer reaction GAE Gallic acid equivalent GM Glucose minimum HAT Hydrogen atom transfer reaction MIC Minimum inhibition concentration MRSA Methicillin sensitive strain of Staphylococcus aureus ORAC O xygen radical ab sorbance capacity SPW Sappanwood or C. sappan heartwood RF5 Water extraction of sappanwood using a 5 hour reflux RF24 Water extraction of sappanwood using a 24 hour reflux RT Water extraction of sappanwood by shaking at room temperature for 6 hours TEAC Trolox equivalent antioxidant capacity TSA Tryptic soy agar TSB Tryptic soy broth TP Total polyphenol VRE Vancomycin resistant enterococci

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial F ulfillment of the Requirements for the Degree of Doctor of Philosophy SAPPANWOOD WATER EXTRACT : EVALUATIO N OF COLOR PROPERTIES, FUNCTIONAL PROPERTIES AND TOXICITY By Valeeratana Kalani Sinsawasdi May 201 2 Chair: Amarat Simonne Major: Food Science and Human Nutrition The r ed color from water extract s of sappanwood (heartwood of Caesalpinia sappan L.) has been used in Thai and Indonesian beverages for hundreds of years. Th e colorant may have potential as a n alternative for synthetic red dyes. However, scientific data on this red colorant is limited. W ater extracts of Caesalpenia sappan prepared from different methods (reflux at 5h (RF 5); 24h (RF 24); room temperature extract at 6 h (RT) ) were evaluated for color, functional properties, and toxicity. Color properties were described by CIE L*a*b* measurements, hue angle, chroma and UV vis spectra Total phenolic (TP) content and antioxidant activities were evaluated by the Folin Ciocalteu colorimetric method, and by the oxygen radical absorbance capaci ty (ORAC) and 2, 2 Diphenyl 1 picrylhydrazyl (DPPH) assays, respectively. Mutagenicity was determined by the Ames assay (with and without metabolic activation) while antimicrobial activity was determined by disc diffusion method. In contrast to other nat urally derived red pigments such as anthocyanins, the color of C. sappan pigments is red at pH 8 12 and appears yellow at lower pH. Brazilei n and brazilin, the compounds responsible for giving red color to the extract, were

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13 detected and identified by a high performance liquid chromatography and mass spectroscopy (LC MS). Total phenolic content and antioxidant activities in RF 24 were not significantly higher than RF5, and thus RF24 was excluded from further evaluation. The extracts (RF5 and RT) showed no mutagenicity. The extracts exhibited antimicrobial activity against common spoilage bacteria including Alcaligenes faecali Bacillus coagulans and Pseudomonas puida at a concentration of 50 mg/mL. The stability of color to heat, fluorescen t light and storage temperature at different pH levels was examined. The h ue of the extract was affected by concentration (0.005 0.4000 mg/mL) but the visual color remained in pink to red shade for pH 8 and 9. Results indicated that the color from sappanwood extr act was more stable at higher pH (9) than at lower pH (7). Overall, results from this current study show the potential for developing sappanwood water extract into a naturally derived red color additive for non acidic food products.

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14 CHAPTER 1 INTROD UCTION Justification A naturally derived r ed colorant that is safe for human consumption is in demand by the food industry. Recent clinical research, whic S ate groups on the use of artificial food colors (McCann and others 2007; Institute of Food Technologists 2011) T he relationship between hyperactivity in children and consumption of synthetic colors was considered inconclusive by experts such as Institute of Food Technologists and the U.S. FDA (Larsen 2008; Gravani 2011) However, t he U.K. Food Standard s Agency, which commissioned the Southampton Study has mandated that food ma nufacturers across European Union to put a health warning on food and drink products containing synthetic dyes starting from 20 July 2010 (Harris 2011; U.K. Food Standard s Agency 2010) Though not mandated, food manufacturers outside the EU have shown increasing interest toward replacing synthetic dyes with more natural alternatives. Gene ral Mills, one of the biggest food compan ies in the world with annual sale s of more than fifteen billion dollars, has long stated the need for color innovation to replace the synthetic color Red 40 ( A llura red) on its company website (General Mills 2011 ) Warner Jenkinson Co., which is a major food color manufacturer, also ac knowledges the consumer demand for natural food coloring (Burrows 2009) Natural colorants have certain limitations su ch as being dull in color, sensitive to pH, and low color strength thus requir ing higher concentration s for food application.

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15 Certain natural colorants such as anthocyanins are susceptible to degradation especially with light and heat, their use is limit ed to low pH food ; also they have a low extraction yield. Furthermore, since they are extracted from plant products such as seeds, skin, or roots, other pro blems include seasonal dependency off flavor, and contamination of insecticides, herbicides, and b acteria can occur (Wrolstad 2004; Griffiths 2005; Castaeda Ovando and others 2009) Exotic sources of natural colorant such as carmine from cochineal insects can be subjected to price variables and public accepta nce (Nachay 2009) Exploring alternative natural colorants with different origins and properties is needed to help food manufacturers meet consumer demands for natural products. Sappanwood or Caesalpinia sa ppan L. is a redwood grown in many parts of Asia Its h istory of us e as a red color ant for beverage s date s back to hundreds of years (Det anand 1975) However, the information on color properties for the C. sappan pigments is currently very limited. Therefore, th is study will evaluate the potential of C. sappan water extract by determining the chromaticity and stability of its pigments, and other benefits such as its antioxidant and antimicrobial activities, and preliminary toxicity Hypothesis The w ater extract of C. sappan heartwood can be developed into a color additive. As it is of plant origin, the pigments obtained may be sensitive to pH, light, heat, and oxygen. The extract may also contain phytochemicals which not only yield red colors, but also have other functional properties such as antimicrobial and antioxidant activities, with low toxicity.

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16 Specific Objectives 1. To review previous published studies and literature on the topics of the C. sappan and its potential benefits. 2. To examine color properties of C. sappan water extracts. 3. To evaluate antioxidant activities of C. sappan water extracts. 4. To obtain preliminary e va luation of toxicity of the C. sappan water extracts in term s of mutagenicity. 5. To evaluate antimic robial activities of the C. sappan water extracts toward food spoilage bacteria 6. To understand the stability of pigments from C. sappan water extracts to pH, light and heat.

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17 CHAPTER 2 LITERATURE REVIEW Caesalpinia Sappan Caesalpinia sappan is know n as East Indian red wood or sappanwood. Although the C. sappan is considered a native plant of India, Malaya (Malay Peninsula) and Sri Lanka, it has been found in many other parts of Asia such as China, Indonesia, Viet Nam, and Thailand. The heartwood of sappanwood was historically valued for both its red pigments and for its medicin al properties throughout Asia. The m edicinal properties of sappanwood were doc umented in the Indian Ayuraveda (Kennedy and others 2004; Kennedy and others 2008) Chinese traditional herbs (Efferth and others 2008) Japanese pharm acopoeia (Na an d others 2001) me dicinal Plants in Vietnam ( WH O, Institute of Materia Medica 1990) and medicinal Thai plants (Det anand 1975) D ocumented pharm acological benefits include activating blood circulation as well as antitumor, antimicrobial, and immunostimulant properties Sappanwood was listed as a natural source of red dye in several scientific references (Defilipps 1998; Vankar 2000; Ferreira and others 2004; Whitney and others 2006; Petroviciu and others 2010; Rosenberg 2008; Cooksey 2009) However, the chromaticity of the extract along with the CIE color measurements ha s not yet been reported. History of Consumption Sappanwood water extract has been used as an ingredient of beverages to quench thirst in Tha iland (Det anand 1975) and in Indonesia (Roesnadi and others 1977; Batubara and others 2009) In Thailand, a red c supposedly contain s sappan wood extract. This concoction ha s been marketed in e than 80 years (Utaitip 2011 ).

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18 Typically a few drops of consumption. In Indonesia the sapp anwood extract is incorporated in several Bir Pletok originated from the Batavian region of Indonesia, and it has a distinctive pink color derived from C. sappan heartwood extract (Batubara and others 20 10 ; Hulupi 2003) as well as o ther ingredients such as ginger and lemongrass. Sappanwood is added to Wedang uwuh drink mainly to impart a red color (Susanto 2010) while other drinks derived from sappanwood are simply called Wedang Secang or Secang drink (Wedang means drink, Secang means sappanwood in Indonesian language). Many recipes for the drinks can be found on World Wide W eb s earch engine s such as Google.com. On the other hand, Jamu is a traditional medicinal drink in Indonesia for health maintenance purposes (Roesnadi and others 1977) and of the m any commercially available Jamu s those c ontaining sappanwood include post partum herbs, which are cl aimed to help reliev e stomach pain after birth ing stimulating blood circulation, and promoting health. Another Jamu called tonic tea was promoted as being able to slow the aging process, improve blood circulation, and increase energy. The other well k nown specie s of the genus Caesalpinia is C. echinata and it is native to South America. C. echinata i s named brazilwood because it also contains red pigment brazilin and the co untry where brazilwood was discovered was later named Since discover ing the red dye from braz ilwood ( C. echinata ) in 1500, it ha s replaced sappanwood ( C. sappan ) as a cheaper source of the red dye. In the middle ages, the heartwood of sappanwood was imported into Europe as a source of red dye (brazilin), which was us ed to dye fabric and as an ink (Irish and Irish

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19 1996 ; Armstrong 1992; Kennedy and others 2004; Berger and Sicker 2009) The primary focus of the current research work is on Caesalpinia sappan or brazili n extracted from C. sappan heartwood. Although the sappanwood ( C. sappan ) extracts have been used in many cultures, limited information on its health effects and epidemiological data are available The objective of this review is to examine the curren t lit erature from 1975 to August 2011 with the goal of covering th e medicinal uses as well as potential benefits (antioxidative, antimicrobial), and detrimental effects of the extract. Furthermore the review will summarize current knowledge related to the chemical composition and current use of the extracts as a food additive with specific focus on the C. s appan heartwood. Color Properties of Sappanwood Extracts Berger and Sicker (2009) proposed that two chromophores exist on a molecule of brazilin (ring A and ring B). W hen those two rings are separated, the molecule does not absorb muc h light in the visible region; h ence, brazilin appears pale yellow or colorless. When the molecule is oxidized, ring B which has a quinone structure b ecome s conjugated with ring A, which abs orbs light in the higher visible wavelength (Figure 2 1). The oxidized brazilin is called brazilein. It is t he quinone structure attached to the aromatic ring of brazilein that acts as a chromophore. A shift in absorbance maxima (bathochromic shift) of brazilein in ethanol from 445 nm to 525 nm occurred when 0.1 N NaOH was added to the solution. Since the color in NaOH solution is red, this color transition is called a red shift. The researchers proposed that the changes in molecular structure of the br azilein molecule were due to deprotonation at the OH group of C3 as illustrated in Figure 2 2

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20 Because brazilein is a natural d ye, it is susceptible to photoo x i dation which affects its chromophore. For the purpose of improving speed for fabric dyeing and minimizing photooxidation, technology such as complexation with transition metals was used. The transition metal or mordants used with the red dye from C. sappan was alum (KAl(SO 4 ) 2 x12H 2 O). Historically not only mordant helps stabilize the color of fab ric, it also helps to make the dye water insoluble. For fabric, less water solubility is desirable since it improves color fastness thus better preserv ing its original color (Vankar 2000) Wongsooksin and others (200 8) investigated the properties of mordant of sappanwood extract and alum, which has been used to dye silk to red color in Thailand. Alum yields Al(III) ion the complex and the researchers proposed a molecular structure of the complex as Al(brazilein) 2 ; t hey reported t he absorption spectra of brazilein as well as the Al brazilein complex. In the experiment, the pH of brazilein solution was controlled at 4.5 without the pH adjustment for the mordant dye solution. The difference that was found in the three ma jor absorption bands suggested that the peak with a maximum absorption at 446 nm was associated with the B ring on brazilein, and the wavelength maxima at 276 nm was associated with the A ring (Figure 2 1) Adding higher concentration s of alum decreases t he absorbance at 446 nm while it increases absorbance at 509 nm. Antioxidant Activities of Sappanwood Extracts High a ntioxidant activities of sappan wood extracts from different geographical locations and from using various solvents including methanol, wat er, petroleum ether, chloroform, and ethyl acetate (Sasaki and others 2007; Pan and others 2004; Badami and others 2003; Sa fitri and others 2003; Na and others 2001) is summarized in Table 2 1.

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21 Using DPPH radical scavenging, Na and others (2001) reported that methanol extract ion of sappanwood (100 g/ml) had 80% higher antioxidative activity (in lipid tocop herol) which ranged from 60 to 80%; they also tested methanol extract of 138 othe r Korean herbal medicine s Yi ngming and others (2004) compared the protective effect of ethyl acetate extracts of sappanwood with BHT against lipid oxidation in peanut oil kept at 60 C for 20 days. Results showed that 0.20% (w/w) of the dried sappanwood ex tract is more efficient than BHT Badami and others (200 3) exami ned the antioxidative activity of s appanwood extracts from India by both in vivo (Nitric oxide) and in vitro assay (DPPH method). The authors used various solvents to extract the C. sappan heartwood, including petroleum ether, chloroform, ethyl acetate, methano l, methanol: water (50:50) and water. The high antioxidant activities in term s of low IC50 w ere reported for ethyl acetate extract, methanol extract, methanol : water extract, and water extract. Petroleum ether extract and chloroform extract, however, sh owed no antioxidative properties. Palasuwan and other s (2005) tested antioxidative activity of water extract of C. s a p pan heartwood from Thailand by ABTS azino bis(3 ethylbenzothiazoline 6sulphonic acid) radical cation decolorization assay. The water extract prepared by boiling in water for 10 minutes (1:20 w/v) showed total antioxidant activity of 9.53 mM/g. The compounds responsible for the antioxidative properties in the aqueous extract s of sappanwood were iso lated by Safitri and Ratu ( 2003 ) and were identified as 1',4' dihydro spiro[benzofuran 3(2H),3' [3H 2]benzopyran] 1',6',6',7' te trol (Figure 2 3) and 3 [[4,5 dihydroxy 2(hydroxymethyl) phenyl] methyl] 2,3 dihydro 3,6 benzofurandiol (Figure 2 4) Both compounds are considered flavonoids.

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22 Table 2 1 Summary of antioxidant activities and benefits of various C. sappan heartwood extr acts in foods Author Country of origin Purpose Extraction Method Application medium Effective concentration Antioxidant activities Benefits in food products Han and Rhee 2005 Republic of Korea n/a Extracted 3x w / 95% MeOH (1:4) by stirring on a hot plate at 40 C for 3 h, filtered. All filtrate combined and dr ied under vac. Stored dried extract in desiccator 12 h, then in air tight glass jar at 20 C. G oat meat and beef patties 0.01% or 0.05% (w/w) Not quantifi ed Antioxidants, retain red color in beef pattie s Palasuwan and others 2005 Thailand Screening of Thai medicinal plants for antioxidant activities Boiled in water at a ratio of 1:20 (w/v) for 10 min Cooled, then centrifuged at 2,500 rpm for 15 minutes, then filte red n/a 1:20 of extract dilution 9.53 mM/g using radical cation decolorization assay n/a Saraya and others 2009 Thailand Antioxidant, antimicrobial and toxicity of sappan water extract Freeze dried, drum dry, and water bath dried of water extraction Thai chi li paste 2, 4, 8 times of the selected max. bacteria MIC DPPH: IC50 at 63.4mcg/ml A fter 3 mos., Total Aerobic Counts were 41.9%, 40.9% and 44.6% respectively Sa saki and others 2007 Japan A ntioxidant and anti inflammatory tests on brazilin, brazilein, sappanchalcone, protosappanins A, B and C Isolated brazilin, brazilein, sappanchalcone, protosappanin A, protosappanin B, and protosappanin C from methanolic extra ction of C. sappan n/a Brazilin completely suppress iNOS gene expression at 100 M Brazilin, brazilein and sappanchalcone significantly inhibit lipopolysaccharide (LPS) induced NO production by J774.1 cell line n/a

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23 Table 2 1. Continued Author Country of origin Purpose Extraction Method Application medium Effective concentration Antioxidant activities Benefits in food products Badami and others 2003 India In vitro and in vivo antioxidant activity of extracts of C. sappan heartwood 10g heartwood powder + 400 DI water, reflux 2 h, cooled, filtered, adjusted final vol to 400 mL HPTLC chloroform: acetone: glacial acetic acid: water = 10:3:2:2 DPPH, IC50 = 6.94 0.96 mcg/mL n/a 10g heartwood powder + 400 DI water microwave d a t 540 W for 20 min DPPH, IC50 = 7.4 0.47 mcg/mL n/a Yingming and others 2004 China Screening of a ntioxi dant properties as PV in peanut oil against BHT 50g ch ips in 500 mL, Soxhlet for 24 h Use rotary evaporator to dry at 60 degree C. Then vacuum d ried at 30 C, 0.07 Mpa. Peanuts oil 0.20% w/w of extract PV = 24.08, compared to PV with BHT, which wa s 26.52 n/a Safitri and others 2003 Indonesia Antioxidant activities of isolated compounds from sappanwood extract Methanol fraction of the intitial water extract was further separated by column chromatography wit h chloroform, ethyl acetate, methanol, and water. Two aromatic compounds were obtained. n/a IC50 of less than 300 g/ml for xanthine oxidase inhibition, superoxide anion radical scavenging, and hydroxyl radical scavenging Inhibitory effect on xanthine oxi dase activity, s c avenging effect on superoxide anion and hydroxyl radicals. Both compo unds were more effective than carotene, and BHT when IC50 values were compared. n/a

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24 Antimicrobial and Probiotic Activities of Sappanwood E xtracts Antimicrobial acti v ities of C. sappan heartwood extracts and their isolated compounds (activ e compounds) have been reported by many researchers Kim and others (2004) examined the antimicrobial activity of C. sappan heartw ood extracts using various solvents (chloroform, n butanol, methanol, and hot water) against the methicillin sensitive strain of Staphylococcus aureus (MRSA). The y found that methanolic extract exhibited the biggest inhibition zone on agar plates, followe d by the extracts from n butanol, chloroform, and aqueous extracts, r espectively A nother study revealed that brazilin was the responsible compound in a methanol extraction of C. sappan heartwood that inhibits the growth of MRSA and vancomycin resistant e nterococci (VRE). This same methanolic extract also show ed inhibitory activity against Streptococcus mutans and Prevotella intermedia which were responsible for dental carries (Xu and Lee 2004) Lim and others ( 2007 ) also evaluated C. sappan extracts as a probiotic factor to inhibit harmful bacteria in the h uman gut and thereby contribute to the growth of desirable gut mi croflora (or probiotic bacteri a). The researchers took the methanol extract of the C. sappan heartwood and further extract ed it with methanol, hexane, chloroform, ethyl acetate, butanol, and water. Each of the secondary extracted fractions were then tested for growth inhibitory responses against both desirable probiotic s i.e. Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus casei and harmful bacteria, i.e. Clostridium perfring ens and Escherichia coli The methanol fraction shows the most preferable activities as it supported the growth of desirable bacteria while suppressed those of the undesirable ones. The methanolic fraction was further separated, and the active compound was identified as 5 hydroxy 1,4 na phtoquinone

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25 The antimicrobial and probiotic activities of C. sappan heartwood extracts shown in these studies revealed its future potential as an antimicrobial agent for medicinal use Medicinal Benefits of Sappanwood Ex tracts Medicinal benefits of C. sappan includ e anti acne (Batubara and others 2010 ) anti inflammatory (Washiyama and others 2009) anti allergenicit y (Yodsaoue and others 2009) anti tumur genesis (Effe rth and others 2008) analge s ic (Hemalatha and others 2007) p rotecti on against oxidation induced cell injury (Bae and others 2005; Cho i and others 1997; Sasaki and othe rs 2007; Shen and others 2007) cardioprotective effects (Zhao and others 2006) diabetes pr evention (Yang and others 2000) and apoptosis induction (Ye and others 2006) Many of these studies w e re conducted to identif y active compounds in C. sappan heartwood pur ified extracts. The potential active compounds were brazilin, brazilein, hematoxylin, and sappanchalcone It was reported that a methanol extraction of C. sappan heartwood yields Brazilin, which is also a natural red dye. Batubara and others (2010 ) isolated brazilin from a methanol extract of C. Sappan and evaluated its antimicrobial, antioxidative, and lipase inhibit ion activities They found that brazilin (Figure 2 1) as well as other C. sappan compounds such as protosappanin A (Figure 2 5) were effective as antimicrobial agent s against Propionibacterium acnes bacteria compared to the co mmon acne medicine tetracycline The authors concluded that brazilin has the poten tial to be an anti acne agent A group of researchers from Japan studied seven compounds is olated from a methanolic extraction o f sappanwood (Washiyama and others 2009) Each compound was tested by an in vitro assay for inhibition of chemical mediators of inflammation, using the J774.1 cell l ine and reported results as IC50 value s (concentration of the

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26 isolated compounds effective to reduce these undesired mediators by 50%). The mediators of inflammation induced the measurement of NO and PGE2 inhibition, as well as the ability to suppress mRN A expression of TNF 6, COX 2, and iNOS. This study also included an in vivo assay for anti inflammatory effect on carrageen a n induced mouse paw edema as % paw edema. The NO synthesized through iNOS gene induces tissue injury. The PGE2 synthesized t hrough the COX 2 gene accelerates inflammation. The TNF ing the synthesis of IL 6 or serotonin, resulting in the activation of T cells and inflammation related cells. Thus the compounds that suppress iNOS, COX 2,TNF 6 gene expression are expected to show an anti inflammatory effect. From the test mentioned, it was found that brazilin inhibited NO production, but almost no inhibition in PGE2. As for sappanchalcone, protosappanin, and protosappan in E, they inhibited both NO and PGE2 production, and suppressed TNF 6, COX 2, and iNOS mRNA expression. When tested in vivo in mice, a crude methanolic extract showed stronger activity than brazilin. Thus, the methanolic extraction of sappanwood c ontained active compounds other than brazilin for the inhibitory effect on mouse paw edema. Structures of the brazilin, protosappanin A, B, C, D, E, and sappanchalcone, are shown in Figure s 2 1 2 5 2 6 2 7 2 8 and 2 9 In another study, C. sappan hea rtwood and roots from Thailand were extracted with dichloromethane and tested for anti allergic activity (Yodsaoue and others 2009) Allergy or hypersensitivity type I is an immune dysfunction. A n a llergic reaction is an immunoglobulin E (IgE) mediated immune response, resulting in a histamine secretion from mast cells and blood basophils. When granules in mast cells or basophils

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27 hexosaminidase is more convenient and has been used instead of detection of histamine for an anti allergy assay. The inhibitory effects were perf ormed on the RBL 2H3 cells and it was foun d that sappanchalcone poss ess ed the most potent antiallergic activity against antigen induced cell degranulation. The sappanchalcone has an inhibitory concentration ( IC 50 ) value as low as 7.6 M, which is 6 fold higher than that of ketot ifen fumarate, a c linical drug w ith IC50 of 47.5 M. The other potent compound identified was the 3 deoxysappanchalcone which has an IC50 of 15.3 M Efferth and others (2008) syst ematic ally screened the bioactivity of natural products that were derived from medicinal plants used in traditional Chinese medicine. Extracts from 76 medicinal plants were evaluated for their abilities to inhibit the growth of tumor cells. Of the 253 ex tracts, 23 extracts from 18 plant species that could reduce cell growth of human CCRF CEM leukemia cells below 20% of untreated cells ( controls ) at a test concentration of 10 g/ml, and C. sappan was among the 18 species identified as having th is potential In the same year, another group of researchers studied the ing sappanwood on the survival rate of late stage (stage IV) gastric cancer patients, and the apoptosis of human gastric cancer cell line (Li and others 2008) They reported that the combination of chemotherapy with oral administration of YWKLF significantly increased the survival of the late stage gastric cancer patients. In addition to sappanwoo d extract, the formula contains five more compone n ts including Panax ginseng. Another study on the analgesic activity of an ethanol extract of sappanwood, with sappanwood from India was reported on Swiss albino mice (Hemalatha and others

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28 2007) The ethanol extract was further fractionated with petroleum ether, diethyl et her, and ethyl acetate for a total of 4 extracts in this study. The test was carried out on Swiss albino mice, which orally received either the extracts or the control for 30 minutes before being induced into pain. The 0.6% v/v solution of acetic acid we re introduced intraperitoneal ly (i.p.), and resulted in pain which was represented by the writhing of the mice. The n umber of writhing in 20 minutes was recorded and it was found that both concentration s of 200 and 400 mg/ml significantly inhibited aceti c acid induced writhing compare d to the effects of aspirin. In the peri od of 2005 2008 many studies were published on th e protective activity of sappan wood an d its extracts on oxidation induced injury to cells. Sasaki and others (2007) reported constitue nts, including brazilin, brazilein, sappanchalcone and 3 types of protosappanin on this oxidation and found that brazilin and sappanchalcone showed higher inhibition s of NO production than other compounds. Choi and others (2007) demonstrated the protective effects of brazilin and sappan wood extracts against tert butylhydroperoxide induced cell death in House Ear Instit ute Organ of Corti1 cells Similar studies on sappanwood and its extracts were also pub lished in other journals (Choi and Ki m 2008; Bae and others 2005; Palasuwan and others 2005) Cardioprotective effects of sappanwood extract were reported. Zhao and others (2006) found brazilein to have cardioactive effects (increased contractility) on heart s isolated from guinea pigs The authors suggested that brazilein had the potential to be developed in to inotropic drugs to modulate the heart condition (strength of heart beats). Xie and others (20 00) reported the evaluation of m ethanolic crude extract of C. sappan along with its isola ted compoun ds Brazilin and hematoxylin on a rat aortic ring to

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29 evaluate their relaxant effects. All three extracts were found to be effective in relaxing the aortic ring which could be supporting evidence to the folk medicine claim that sappanwood promo tes blood circulation Brazilin was also tested for its ability to enhance cellular immune responses in type I diabetic mice. It was found that brazilin increased the responsiveness of immune cells, and the authors sugg ested that brazilin might have the p otential to prevent type I diabetes that is induced by bacterial or fungal infections (Yang and others 2000) The oxidized form of brazilin is called brazilein, this compound also has been reported to have some pha rmacologic potential such as increasing immunity, improving cardiovascular health acting as an anti inflammatory as well as being a relaxant. Ye and others (2006) reported that methanolic extract of C. sappan hea rtwood has immunocompetence effects including the property of inducing apoptosis of mice spleen lymphocytes and the ability to inhibit lymphocyte proliferation. The author s suggested that the active compound in this extract was brazilein. Brazilein was f ound to have anti inflammatory effect and thus, may provide some protection to the brain against ischemia/reperfusion injury (Shen and others 2007) In a study on rat thor acic aorta, effectiveness of 100 M of brazilein was reported as 116% of 20 mM caffeine in inducing the contraction (Shen and others 2008) Chemical Compounds Isolated from C. sappan H ea r twood Chemical compounds in sappanwood have been identified and reported by many authors Techniques used for separation and identification include solvent extraction, X Ray, NMR and MS. A group of Japanese researchers led by Toshihiro Nohara published several reports in the Saitoh and others ( 1986) examined the methanol extraction of C.

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30 sappan heartwood for antihypercholesteremic activity and identified 6 aromatic compounds includin g 3 benzylchroman derivatives proposed to be precursor s of brazilin biosynthesis. The complete molecular structures of these compounds were not determined. Shimokawa and others (1985) used spectroscopy to identif y compounds in sappanwood and they reported two aromatic compounds which they named caesalpins J and P. In another study, Miyahara and others (1986) used X ray analysis to identify an interesting compound from a methanolic extract, and which was called It was thought to be derived from the 3 benzylchroman derivatives id entified previously by Saitoh and others (1986) Fuke and others (1985) detected the responsible compounds for hypercholoesteremia activity of C. sappan heartwood extract and identif ied 2 aromatic compounds They simply call ed them compound 2 and 3, with compound 1 being brazilin. Nagai and others ( 1984 1986) reported the isolation of protosappanins A (I, II, and III) B, and C (Figure 2 5 and 2 6 ) Protosappanin A (I) was also identified by Batubara and others (20 10 ) and Washiyama and ot hers (2009). Protosappanin C was also identified by Washiyama and others (2009) Namikoshi and others (1987) worked with several other researchers to identify compound s from sappanwood extract s They published 5 research articles in 1987 and id entif ied a total of 7 new compounds from sappanwood along with other 15 known compounds that were separated for the first time This group reported structures and categorized a novel class of homoisoflavonoids which contain a 3,4 dihydroxy homoisoflavan s tructure as illustrated in Figures 2 10

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31 Additional reports on the separation and identification of sappanwood extract were mostly after 2000. Yang and others (2002) used ethanol extract ion of sappanwood to isolate a new compound. The ethanol extraction that is soluble in ethyl acetate was furth er separated by repeated column chromatography over silica gel. The compound isolated was named lactone brazillide A (Figure 2 11 ) The molecule is highly oxygenated and i s suspected to be a product of aut oxidation for brazilin Zhao and others ( 2008) worked on an ethanolic extract of sappan wood and reported a new homoisoflavan, which ha s a brown colo r The compound was identified trihydroxy 3 benzyl 2H chromene (Figure 2 12 ). Fu and others (2008) reported 3 new compounds, together with those previously identified These compounds are 3 benzylchroman derivatives. According to their report, the phenolic compounds in sappanwood extract are grouped into f our subtypes by structure, i.e. brazilin, chalcone, protosappanin, and homisoflavonoid. Sappanwood Extract as a Food Additive This section summarizes re ports on applications of sappan wood extract as a food additive, benefits include using it as a red color ant, food antioxidant, and food preservative. Indrayan and Guleria ( 2001) reported the physical appearance and yields of dyes extracted from sappanwood and proposed its use as food colorants. Depending on solvents, i. e. water, methanol, ethanol, aqueous NaOH, colors ranged from orange red, orange, to red violet with yields ran ging from 0.838% to 5.025% (Guleria and others 1997) ; however, this study did not identify the active compounds. Another study by Badami and others (2007) examined the effective ness of a red dye extracted from sappanwood using water and microwave heating The researchers found that the extraction assisted by microwave heating at 540 watt for 20 minutes increased the yield

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32 of the red dye by almost 14% but they did not identify t he chemical compound ( s ) responsible for the red color. However, they confirmed that the compounds from microwave assisted extraction were the same as the traditional hot water extraction since they both had the same UV spectrum, Rf o n TLC, and peak area o n HPTLC. In addition to the red color, the microwave assisted water extract also exhibited the same antioxidant activities as demonstrated using DPPH and nitric oxide methods. To date, only three articles have been published regarding the application of sappanwood extract in food one is on antioxidative while the other two are for antimicrobial applications. The antioxidative properties of the ethanolic extract of sappan wood in beef patties were studied by Han and Rhee (2005 ). Their results show that at a concentration as low as 0.01%, the sappanwood ethanolic extract was effective in preventing lipid o xidation in beef patties Saraya and others (2009) reported the highest antimicrobial activity in a freeze dried water extract of sappanwood compared to oven drying and water bath drying methods The freeze dried extract was effective against Escherichia coli Staphylococcus aureus and Salmonella Typhimurium as determined by the Minimum Inhibitory Concentration (MIC) below a 500 g/mL. When applying to chili paste, a popular local product, the sappanwood extract showed protection against bacterial growth for up to 6 months. The extract had no major influence on fun gal growth. In another study regarding using sappanwood extract for food preservation, the antimicrobial activity was evaluated using an agar well diffusion method against common spoilage bacte ria and yeast in coconut milk. The sappanwood extract showed protection based on zone of inhibition (mm) against several Bacillus lichniformis and Klebsiella pneumoniae strains, and Trichosporon mucoides The

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33 extract, however, showed no activity against yeast (Phattayakorn and Wanchaitanawong 2009) Conclusion Extracts of C. sappan heartwood, or the so called sappanwood, have been used in many cul tures. C. sappan heartwood extracts from various solvents have been documented to have potential as natural food additives, as a health promoting beverage, and also as medicinal drugs. The food additive potential of these extracts includes use as red foo d colorants, antioxidants and antimicrobials to help prolong shelf life and promote food safety in various food products. B razilin is an important compound as i t is responsible for many of the functional properties of the sappanwood extract, as well as b eing a precursor of the red colorant, bra zilein. A few of the many possible active chemical compounds in sappanwood extracts promoting these applications have been identified.

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34 Figure 2 1. Molecular structures of brazilin (left) and brazilein (ri ght). Figure 2 2 Proposed mechanism of red shift of brazilein when 0.1N NaOH was added Figure 2 3. 1',4' dihydro spiro[benzofuran 3(2H),3' [3H 2]benzopyran] 1',6',6',7' tetrol A A B B

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35 Figure 2 4. 3 [[4,5 dihydroxy 2(hydroxymethyl) phenyl] methyl] 2,3 d ihydro 3,6 benzofurandiol Protosappan A (I) R 1 = H R 2 = O Protosappan A (I I ) R 1 = CH 3 R 2 = O Protosappan A (I II ) R 1 = H R 2 = H, OH Figure 2 5. Protosappanin A Protosappanin B, R 1 = CH 2 OH R 2 = OH Protosappanin C, R 1 = CHO R 2 = OH Figure 2 6. Protosappanin B and protosappanin C

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36 Figure 2 7. Protosappanin D Figure 2 8. Protosappanin E Figure 2 9. Sappanchalcone

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37 Figure 2 10. 7 hydroxy 3 hydroxybenzylidene) chroman 4 one Figure 2 11. Brazi lide A Figure 2 trihydroxy 3 benzyl 2 H chromene

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38 CHAPTER 3 ANTIOXIDANT CAPACITIES AND COLOR CHARACTERISTICS OF SAPPANWOOD WATER EXTRACTS Background Overview T he solvation extraction involves a partition process in which chemical compounds in the solid matrix are transferred to the extractant. The process begins with the sorption and capillarity of the solvent causing sample particles to swell, and then any soluble components diffusing from the solids into the liquid. The factors that cont ribute to efficacy of the solid/liquid extraction process include type and volume of solvent, pH, temperature, particle size of solid, and number of extraction steps (Self 2005) For a complex sample such as heartwood, ext raction at high temperature s such as with a boiling solvent, can be less desirable as it contributes to non active compounds being forced into solution (Berger and Sicker 2 009 ) Boiling has been used for the extraction of color compounds from sappanwood for food use (Det anand 1975) This extraction method is considered a solid/liquid extraction type with sappanwood as a solid and hot water as a solvent ( liquid). Color is very important for foods and beverages because it is the first attribute that consumers see when they look at a food product. Color instruments can be used to measure color as opposed to a visual inspection, which can be subjective. Of the many color measurement standards the CIE La*b* system ( International Commission on Illumination, Vienna) is widely used in the U.S. food industry (Wrolstad and others 2005; Wallace and Giusti 2008) The CIE e stablished a standard for the light source used in color measurement s such as D 65 for the average daylight and A for the incandescent. Color can be represented in terms of hue (color), lightness (brightness or value), and chroma (saturation). Color as w e name it is represented by hue which can be

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39 calculated from redness (a* value) and yellowness (b* value) as arctan of b*/a* Hue is represented by the angle from 0 to 360 with 0 as red, 90 as yellow, 180 as green and 270 as blue. Color can be bright, midtone, or dark and can be represented by L* value (lightness) with 100 means absolute white and 0 means absolute black. Apart from hue and lightness, another value independent to neither hue or L* is Chroma or saturation. High chroma value indicat es higher saturation of color (Wrolstad and others 2005). Antioxidant polyphenolic compounds from plants, in particular, fruits and vegetab les, have been shown to have the s e health promoting properties A ntioxidant activities and total phenolic aci d content may be used as one of the criteria for screening and selecting the right extractions of sappanwood water extract in addition to measuring color intensity. Most antioxidant activity measuring methods share the same principle, i.e. generating free radical species, detecting an end point, and then compar ing it to the protecti on of the sample (Rice Evans and others 1996) Depending on mechanisms, antioxidant activities are var ied (Tsao and Deng 2004) Many in vitro and in vivo models have been used to evaluate the antioxidant activities of polyphenols. The ox ygen radical absorption capacity (ORAC) method and Trolox equivalent antioxidant capacity (TEAC) are based on the ability of the antioxidant to neutralize peroxyl radical. According to Huang and others (2005), the ORAC mechanism is based on the hydrogen a tom transfer (HAT) reaction. The other type of antioxidant capacity measuring assay is based on the ability of antioxidants in to reduce oxidant through electron transfer (ET) reaction s The assays that are based on ET reaction include 2,2 di(4 tertoctyl phenyl) 1

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40 picrylhydrazyl (DPPH) and the total phenols assay by Folin Ciocalteu reagent. DPPH can be used to evaluate the antioxidant capacity of fruit and vegetable juices or extracts. The ORAC is a common assay for antioxidant activity evaluation of nat urally occurring phytochemicals including botanical extracts (Bykbalci and El 2008; Fernandez Panchon and others 2008; Talegawkar and others 2009) The objectives of this study are to evaluate total polyphenol content, antioxidants activities and chromaticity of sappanwood water extracts, both at boiling (reflux) and at room temperatures (shaking) Materials and Methods Raw Materials and Chemicals Sappanwood was purchased from Jao Krom Poe the oldest and most reputable Thai herb pharmacy in Bangkok, Thailand (Usuparatana 1997) Methanol, Folin Ciocalteu Reagent, gallic acid, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic dehydrate, and sodium carbonate were purchased from Fisher Scientific C o., (Pittsbur gh, PA, U.S.A. ). 2,2 D iphenyl 1 picrylhydrazyl (DPPH), 6 hydroxy 2,5,7,8 tetrame thylchroman 2 carboxylic acid (Trolox), and fluorescein (free acid) were purchased from Sigma Aldrich ( St. Louis, MO U.S.A. ) Azobis(2 amidinopropane) di hydrochloride (AAPH) was purchased from W ako Chemicals (Richmond, VA, U.S.A. ) E xtracts Preparation Size reduction of the heartwood chips The pieces of sappan heartwood chips were reduced to 2 mm or less with Wiley Mill as shown in Figure 3 1 and Figure 3 2 (Thomas Wiley Lab Mill Model 4 ; Swedesboro, NJ, U.S.A. ). Size reduction of the sappanwood chips to sappanwood

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41 powder is illustrated in Figure 3 3. The sappanwood powder was manually shaken in large plastic bag for 10 minutes to homogenize, then transf erred to an airtight container and kept at 10 C in a freezer Water extraction of sappanwood The water e xtraction of sappanwood was done with a hot water extraction and cold water extraction, and each extraction was performed in triplicate. The f ir st tr eatment was a reflux of 5.0 g of sappanwood powder in 200 mL of deioniz ed water for 24 hours (RF24). The s econd treatment was similar to the first one, but the reflux was only 5 hours (RF5). For the cold water extraction (RT), 3.0 g of sappanwood powder was added to the Erlenmeyer flask with 100 mL of deionized water and shaken for 6 hours on a n Excella E1 Platform shaker (New Brunswick Scientific : Edison NJ U.S.A. ). Note: because there was no previous study on the cold water extraction of sappanwood, a preliminary study in house was conducted at extraction times of 2, 4, 5, 6, 7 and 8 hours in triplicate. The h ighest absorbance at 445 nm was observed at 6 hours of extraction by shaking with 3.00 grams of sappanwood in 100 mL of water at room temperatu re. Additional det ails of the study can be found i n Appendix A. After extraction e xtracts were centrifuge d with Beckman Coulter, Allegra X 15R (Brea, CA, U.S.A.) for 5 minutes at 1738 x g ( 4000 rpm ) then filtered through Whatman #1 filter paper. The filtered extracts were freeze dried in a freeze dryer (Free ze dryer 5, Labconco Kansas City, MO, U.S.A. ). The f reeze dried extract s were identified as RF24, RF5, and RT, and were stored at 20 C until subsequent analyses on total phenolic content, anti oxidant capacities, chromaticity, and brazilin/ brazilein were analyzed

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42 Folin Ciocalteu Assay for T otal P henolic C ontent Freeze dried extracts ( 0.01 g ) were reconstituted with 1 .0 mL of methanol. Gallic acid in methanol at concentrations of 0, 75, 150, 300, 450, and 600 mg/L were used as standards for the calibration curve. E ach test tube contain ed 1 mL of 10% of 2 N Folin Ciocalteu solution in water, 100 L of sample solutions ; and 1.0 mL of 15% sodium carbonate. After vortexing, tubes were incubate d for 30 minutes at room temperature. A microplate reader (Molecular Devices, SPECTRA max 190, with softmax pro software; Sunnyvale, CA, U.S.A.) was used to read absorbance at 765 nm and compare results against calibration curve s of gallic acid. Results a re reported as mg/L gallic acid equivalent. The assay was done in triplicate. Antioxidant Activity DPPH Radical Scavenging Activity Sample solutions were prepared by dissolving 0.01 g of freeze dried extracts in 1 mL of methanol and the final volume was adjusted to 10 mL in volumetric flask with deionized water. Trolox in methanol with concentrations of 0, 200, 400, 600, 800, and 1000 M were used as s tandard solut ion used as calibration standards. A w orking solution of DPPH was prepared by diluting DPP H with methanol to the initial absorbance of 0.9 1.0 at 515 nm. The assay was performed by adding 950 L of D PPH solution and 50 L of an aliquot of samples or standards, then vortex ed to mix. After incubation at room temperature for 1 hour, absorban ce at 515 nm was measured by a microplate reader (Molecular Devices, SPECTRA max 190, with softmax pro software; Sunnyvale, CA, U.S.A.) T he assay was done in triplicate and results are reported as M of Trolox equivalent.

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43 Oxygen Radical Absorbance Capaci ty (ORAC) Solution s of Trolox in phosphate buffer pH 7.4 w ere prepared at concentration s of 0, 5, 10, 15, 20, and 25 M to be used as the standard reference s for the calibration purpose s The f reeze dried sappanwood water extracts were reconstitute d with 10% methanol in deionized water, at a dilution of 1:100 and 1:1000. The experiment was performed in a 96 well plate with a microplate reader (SPECTRA max Gemini XPS, Molecular Devices, Sunnyvale, CA, U.S.A. ). In each well, 50 L of Trolo x or sample solut ions were added; for a blank, phosphate buffer was used in place of the sample. Then, 100 L of 20 nM Fluorescein was added into each well, and the plate was mixed for 3 minutes a nd incubate d for 7 minutes at 37 C. To start the reaction, 50 L of 140 mM AAPH in the phosphate buffer was added into each well immediately using an 8 well multi channel pipet. Fluorescence of each well was measured every minute for 40 minute s then the differences of areas under the curve of fluorescence and time was calculate d by the software (SoftmaxPro5 Molecular Devices; Sunnyvale, CA, U.S.A. ) and expressed as mole Trolox equivalents per mg of the freeze dried samples. Chromaticity of Sappanwood Water Extract Two of the freeze dried hot water extractions, i.e. the 24 hour reflux (R F24) and 5 hour reflux (RF5) were weighed to 0.10g and diluted with deionized water 100 mL in 100 x 16 mm test tubes. Then, 3.0 mL of the solution was added to 12.0 mL of phosphate buffer pH 2 12 For the cold water extraction (RT), similar pre par ation was performed but only 0.0400 g of the freeze dried SPWE was used. This concentration is to accommodate similar absorbance max at pH = 9. Details on the buffer preparation can be found in Table 3 1

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44 Brazil e in Analysis (HPLC DAD ESI MS n Analysis) Brazilein was analyzed by HPLC using an Agilent 1200 SERIES HPLC system (Agilent, Palo Alto, CA U.S.A. ), equipped with an autosampler/ injector and diode array detector. Compounds were then separated on a SB C 18 Zorbax Stablebond Analytical column (4.6 mm x 250 mm, 5 M, Agilent Technologies; Rising Sun, MD U.S.A. ) The m obile phases consisted of 0.5% formic acid in H 2 O (phase A) and methanol (phase B). The flow rate was 1 mL/min. The UV vis spectra were scanned from 220 to 650 nm on a diode array de tector with detection wavelengths of 445 and 556 nm. A l inear gradient was used as follows : 0% to 5% B from 0 to 2 min, 5% to 30% B from 2 to 10 min, 30% to 40% B from 10 to 50 min, 40% to 85% B from 50 to 60 min, 85% to 95% B from 65 to 70 min, and 95% to 0% B from 70 to 75 min ; followed by a re equilibration of the column for 5 min before the next sample analysis. Then an Electrospray ionization mass spectrometry (ESI MS) interfaced with the HPLC system was performed with an HCT ion trap mass spectromete r (Bruker Daltonics; Billerica, MA U.S.A. ). An e lectrospray ionization was performed at both positive and negative mode s during the same run using a nebulizer 45 psi, drying gas at 11.0 L/min, a dry temperature of 350 C, and an ion trap for scanning ran ging from m/z 100.0 to 2200.0. Statistical Analysis Each treatment condition was repeated in triplicate. An a nalysis of variance Studentized (HSD) Range test (p < 0.05) were performed in order to evaluate the di fferences in total phenolic content and antioxidant activit i es between extraction times, temperatures, and treatment s; SAS 9.2 statistical software was used (SAS Institute Inc., Cary, NC, U S A ) An e xample of the SAS output is shown i n Appendix B.

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45 Resul ts and Discussion Total Phenolic Content and Antioxidant Capacities T otal phenolic content as measured by the Folin Ciocalteu assay from different extraction methods are in Table 3 2 i.e. RF24, RF5, and RT. Total polyphenol content in RF24 was 17.15 GAE/ g of SPW (mg gallic acid equivalent per g of sappanwood). The amount is significantly lower than that which resulted from less extraction time (5 hour extraction, RF5) which wa s 27.92 GAE/g of SPW. The antioxidant capacity of RF 24, as measured by the DP PH method was also significantly lower than the antioxidant capacity of RF5. However, the antioxidant capacit ies determined by the ORAC assay s of both samples wer e not significantly different. This may be because the prolonged heat exposure such as the 24 hour reflux used with RF 24 caused more degradation of certain antioxidative compounds than shorter extraction times Since the DPPH assay has a different antioxidant principle than the ORAC assay, it is likely that the antioxidative compounds that we re active in the hydrogen atom transfer reaction and detected in the ORAC assay were not as sensitive to heat as the antioxidants that based their activity on an electron transfer a type of transfer which is detected by the DPPH assay (Huang and others 2005) The cold water extract (RT) was found to have the highest total polyphenol content compared to the hot water extractions (RF5 and RF24) However, the antioxidant capacities of RT as measured by the DPPH and ORAC assay s were not significantly different from the antioxidant capacities of RF5. A possible reason why the polyphenol content was high when the antioxidant capacity remained the same is because although there is a higher content of heat sensitive po lyphenols in RT, these heat sensitive compounds ar e not antioxida nts Moreover, heat applied during the extract ion could

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46 contribute to more of the compounds with no antioxidative properties leaching out to the extract. Since the antioxidant capacities in this experim ent were calculated against the dried weight of the extract, it is possible that the weight proportion of the antioxidative compounds compared to the non active compounds of the heated extracts are less than the weight proportion to the non he at treated extract s Thus, the room temperature extraction may be more desirable in term s of antioxidative propert ies as less energy is required for the comparable amount of antioxidative compounds obtained. The h igher antioxidat ive capacity of sappanwo od extracts may be a potential indicat or of potential health benefits. An antioxidant activity can serve as another criterion for choosing the method of extra ction when using sappanwood as a food additive Chromaticity of S appanwood W ater E xtract Brazi le in the compound that is responsible for the red color in sappanwood extract, wa s detected but not quantified in all samples (RF24, RF5, RT). Then, m ass spectrum in positive ion mode of brazilein is shown in Figure 3 4 The spectrum displays the ion [M+H ] + with m/z = 285. In addition, further fragmentation also yields m/z = 175 which agreed with the fragmentation suggested earlier by Hulme and others ( 2005) Freeze dried sappanwood water extr acts were reconstituted by diluting them with an aqueous pH 9 buffer to achieve a chroma value (color intensity) of approximately 30. As shown in Table 3 3 RF24 and RF5 at the same concentration of 0.22 mg/mL contribute to a chroma value of at least 29 w hile RT at 0.08 mg/mL had a chroma value of more than 34 suggest ing that higher amounts of colorant compounds exist in RT compared to RF24 and RF5 at the same pH (pH = 9). The visual colors of the extracts

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47 are shown in Figure 3 5 The pH of 9 was chosen because of the highest a*, chroma values and highest absorbance at max compared to others in the range of 2 12. The higher color saturation at lower concentration s of the extract is desirable as less colorant is required to achieve the same result, thus RT is also preferable on this criteria. The result agreed with the earlier analysis, which suggests that higher heat applied during extraction did not contribute to higher antioxidant activity. Thus, RF5 along with RT were chosen for further evaluation The c olor measurement was performed using CIE in term of redn ess (a*) and hue angle, and it was found that the redness (a*) increased sharply when the pH was 6 and higher (Figure 3 6 ). This trend was applied to all extracts (RF24, RF5, and RT). Since color is three dimensional the hue angle is taken into consider ation as plotted in Figure 3 7 The plot show s that at lower pH levels extracts were more yellow while solutions at pH levels of 7 and over were red to red orange. This property of changing to red color at higher pH is the opposite of the well known nat ural red pigment, anthocyanins, which change to blue at higher pH levels (Wrolstad 2004) Thus, this property provides a possibility of applying a natural colorant to food products with higher pH values To date no other natural colorants provide red color to low acidic food products Therefore, sappanwood extract s may offer a possibility to replace synthetic color ing in these food products. The UV vis spectra of the RT at p H 2 12 are shown in Figure 3 8 A s hift of spectral band (bathochromic effect) is found at pH 7 and above. The absorbance maxima was found at 539 nm ; this is different from the previous report of 525 nm by Berger and Sicker (2009). However the plot of the absorbance at max shows a similar

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48 trend, i.e. higher absorption in the visible region when the pH of the solution is 7 and over (Figure 3 9 ) Conclusion It was found that the hot water extraction at shorter reflux time (RF5) along with the cold water extraction (RT) we re chosen for subsequent analysis due to their high antioxidant activity and high color saturation. Bathochromic shift was found on U V Vis spectra to the absorption maxima of 539 at pH 7 and over, which agreed with the increase in redness and hue angle to ward red color when the pH was higher. This property can offer a n advantage over the popular natural red colorant, anthocyanins, the application of which is limited to only low acid food s

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49 Figure 3 1. Wiley Mill (Thomas Wiley Lab Mill Model 4). Figur e 3 2. Metal screen with 2 mm diameter attached to the Wiley Mill (Thomas Wiley Lab Mill Model 4). Figure 3 3. Sappanwood powder ground from sappanwood chips.

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50 Table 3 1 Buffer preparation for c hromaticity test ing pH Volume (mL) Reference 0.2 M KCl 0.2 M HCl 0.1 M Citric acid 0.2 M dibasic sodium phosphate 0.05 M Disodium hydrogen phosphate 0.2 M monobasic sodium phosphate 0.2 M Glycine 0.1 M NaOH 0.2 M NaOH Final volume 2 50 6.5 100 Haynes 2011 3 39.8 10.2 100 Stoll and Blanchard 2009 4 30.7 19.3 100 5 24.3 25.7 100 6 17.9 32.1 100 7 6.5 43.6 100 8 94.7 5.3 200 9 50 8.8 200 10 50 32 200 11 50 4.1 100 Haynes 2011 12 50 26.9 100 Table 3 2 Total polyphenol content and antioxidant activities of sappanwood water extract s (RF5, RF24, RT) Extraction method Total Polyphenol (mg GAE/ g of SPW chips) DPPH (mM Trolox equ i valent/g of SPW chip s) ORAC (mcM Trolox/g of SPW chips) Reflux 5 hrs 27.92 b 1.68 64.23 a 6.59 12.46 a 0.90 Reflux 24 hrs 17.15 c 1.06 33.88 b 2.24 13.81 a 0.74 Shake 6 hrs at room temp 49.91 a 0.42 76.12 a 10.61 14.21 a 0.48 Values expressed are me an SD of three experiments. Data represents the mean of n=9. Values with similar

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51 Figure 3 4 Positive ion electrospray product ion mass spectra of brazilein. Brazilei n is detected in the extract of a sappanwood sample using HPLC MS 2 showing ion with m/z = 285 in positive ion mode, further fragmentation also yields m/z = 175. Molecular structure s of fragments agree with the propose d by Hulme and others (2005) Table 3 3 Chroma of sappanwood water extracts at pH 9 Extracts Concentration (mg/mL) Chroma at pH = 9 RF5 0.22 30.49 RF24 0.22 29.55 RT 0.08 34.22

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52 Figure 3 5 Visual colors of aqueous solution of sappanwood extracts (RF5, RF24, RT ) at pH 9. P hotos from top to bottom are RF5, RF24, and RT. Figure 3 6 Redness of sappanwood water extract s represented by mean a* value ( n = 3 ). -10.00 -5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 0 2 4 6 8 10 12 more yellow < -a* -> more red pH RF24 RF5 RT

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53 Figure 3 7 Chromaticity as a*, b* and hue ang les of SPWE The h ue a ngle s of SPWE pH 2 5 were low in a* but hig h in b* (data points are in the brown circle). The h ue angle s of SPWE pH 7 12 were high in a* (data points are in the red circle. The hu e angle s of SPWE at pH 6 are in the yellow circle.

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54 Figure 3 8 UV vis spectra of sappanwood (RT) extract showing bat hochromic shift at pH 7 and higher. Figure 3 9 Absorbance of sappanwood extracts RT at 525 and 535 nm in buffer solutions pH 2 12. -0.4000 -0.2000 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 1.2000 1.4000 250 300 350 400 450 500 550 600 650 700 Wavelength (nm) pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 pH 12 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 1.2000 0 2 4 6 8 10 12 Absorbance pH Abs at 525 nm Abs at 539 nm

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55 CHAPTER 4 AMES MUTAGENICITY AS SAY AND ANTIMICROBIA L ACTIVITY OF SAPPAN WOOD WATER EXTRACTS Background Overview In ord er to assess the potential of developing sappanwood water extract as a food additive, toxicological testing is essential. The Office of Food Additive Safety, U.S. FDA, has recommended several toxicological tests as a guidance for the food industry (C enter for Food Safety and Applied Nutrition/ U.S. Department of Health and Human Services 2006) Genetic toxicity tests, such as the bacterial reverse mutation test are common tests recommended for all chemicals, including those with low, intermediate and high concern levels. The bacterial reverse mutation test or Ames mutagenicity assay uses different strains of Salmonella Typhimurium which were mutated for different sensitivity towards different types of DNA damaging chemical mutagens. Unlike regu lar S almonella these mutants cannot synthesize biotin from histidine. Because biotin is essential for their growth, these S almonella strains used for the Ames test cannot grow in an environment with limited availability of biotin unless they undergo a rev erse mutation. The reverse mutation is caused by an exposure to certain mutagens which can restore their ability to synthesize biotin from histidine (Maron and Ames 1983) In the assay, a glucose minimal agar plate with top agar containing a minute amount of histidine was used to create the environment with limited histidine and biotin for the Ames mutagenicity test. Since sappanwood extract may contain xenobiotics (organic compounds not normally produced in the body), their metabolites too should be analyzed because they may be to xic while the original compound is not. In order to enable the bacterial assay to metabolize the test chemicals via cytochrome P450 the same way as mammals, a

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56 metabolic activation system such as rat liver homogenate or S 9 microsomal fraction containing cytochrome P450 is added to the test system (Maron and Ames 1983; Mortelmans and Zeiger 2000) The S 9 fraction used in the Ames test is a supernatant of liver homogenate of rat centrifuged at 9000 x g for 20 min utes, which contains cytochrome P450. The cytochrome P450 is a group of major enzymes involved in the metabolism of xenobiotics in mammals (Guengerich 2007) For general screening, a tier approach is recommended. In this approach, S Typhimurium strains TA98 and TA 100, both with and without metabolic activation, were recommended to be used in the initial step. Results are represented as mean of revertant colonies per plate standard deviation. While t here are many appr oaches on how to determine mutagenicity t he setting of fold increase as a cut off point is widely used ; and usually when there is a 2 3 fold increase (in the numbers of colonies) from negative control, the extract is considered mutagenic (Mortelmans and Zeiger 2000) The a ntimicrobial property of sappanwood extract was investigated in several studies. The ant i microbial susceptibility spectrum includes those pathogens that are of clinical importance, and tho se food spoilage bacteria and fungi. For its medicinal use, (Xu and Lee 2004) studied the principle antibacterial properties of sappanwood, which included the identification of responsible compound, the spectrum of antibacterial activity as we ll as the mode of action. In this study, the researchers first evaluated different fractions of sappanwood extract against methicillin resistant Staphylococcus aureus ( MRSA ) 595445 and vancomycin resistant enterococci ( VRE ) and found that the methanolic extract was slightly more effective than the aqueous extract. When the methanolic extract was further fracti onated with other solvents, such as hexane, ether,

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57 butanol and water, the ether fraction was shown to have the highest antimicrobial activity. Thi s fraction was then further separated with silica gel column chromatography and thin layer silica gel chromatography. The responsible compound in the ether fraction was identified as b rasilin ( with the same molecular structure as brazilin on this current report) by 1 H NMR spectrum. When it was tested against fourteen bacterial pathogen s it was found that brazilin had high antimicrobial activity against Streptococcus pyogenes (Group A Strep) M1 and Streptococcus agalactiae (Group B Strep) A909 with the mi nimal inhibitory concentration (MIC) as low as 4 g/mL. In the attempt to understand the mechanism of antibacterial action, a radiolabel incorporation assay was used to evaluate the effect of brazilin on DNA and protein synthesis by MRSA using dimethyl su lfoxide ( DMSO ) as a negative control. The low level of incorporated radiolabel thymidine and serine of brazilin samples showed that the antibacterial effect resulted from the inhibition of DNA and protein synthesis. In addition, brazilin did not show cyt otoxicity to Vero cells up to 1 mg/mL. Thus, these researchers concluded that brazilin has the potential to be developed into an antibiotic. Another group of researchers from Korea (Kim and others 2004) also investigated the antimicrobial activity of sappanwood extract against several strains of MRSA using a disc diffusion method and the researcher s also determined the MICs of the extracts. Similar to the study by Xu and Lee (2004), the methanolic extract of sappanwood was found to have higher antimicrobial activity, but active compounds were not identified in the study. Lim and others (2007) tested the antimicrobial properties of four isolated compounds from sappanwood against intestinal bacteria The intestinal bacterial

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58 strains tested were pathogens as well as pr o biotics, i.e. Bifido bacterium bifidum ATCC 29521, Bifidobacterium breve ATCC 15700, Clostridium perfringens ATCC 13124, Escherichia coli ATCC 11775, and Lactobacillus casei ATCC 27216. It was concluded that the 5 hydroxy 1,4 naphthoquinone (Figure 4 1) had an inhibitory effe ct selectively on pathogens such as C. perfringens and less inhibitory effects on pr o biotic bacteria. For food preservation using sappanwood extract, two studies were documented. First, Phattayakorn and Wanchaitanawong ( 2009) used a disc diffusion method to test 25 Thai herb extracts against 11 strains of coconut milk spoilage microorganisms. They found that methanoli c extract of sappanwood had an antimicrobial effect on some bacteria such as Bacillus licheniformis Klebsiella pneumonia and Trichosporon mucoides, but they did not identify active antimicrobial compounds. The second study evaluated the antimicrobial act ivity of sappanwood extract against E. coli, S. aureus, S. Typhimurium and C. albicans (Saraya and others 2009) In this study, the investigato rs found that freeze dried powder of sappanwood extract showed higher antimicrobial activity than drum or water bath drying methods. The freeze dried powder of the sappanwood was selected for its high antimicrobial activity for further evaluation. When the freeze dried extract was applied as a preservative to chili paste, a popular Thai food ingredient, t here was no inhibitory effect against fungal growth, but the extract did show inhibition against microbial growth (total aerobic plate count) for up to 6 months. The objectives of this study are to obtain preliminary toxicity evaluation of sappanwood water extracts (both at boiling RF5, and at room temperature RT) in term of mutagenicity, as well as antimicrobial activities against spoilage bacteria The Ames

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59 mutagenicity assay will be used for the toxicity evaluation, and a disc diffusion method will be used to evaluate the antimicrobial activity. The antimicrobial results obtained can help to determine additional functional benefits of sappanwood as a food preservative, while the Ames mutagenicity test can serve as additional evidence of its toxicity. Materials and Methods Sappanwood Extracts Preparation Two sappanwood extracts, RF5 and RT were used in Ames mutagenicity and antimicrobial assays. The ex traction method was described in detail earlier in Chapter 3. Briefly RF5 wa s a hot water extraction of sappanwood using a reflux apparatus for 5 hours, and RT was an extraction at room temperature by shaking for 6 hours. Each of the extracts was dilute d to 500, 5000, 25000, 33330, and 50000 g/mL which is the weight of the freeze dried extract to ehe volume of sterile water with sterile deionized water These concentrations contribute to the concentration per plate of 50 to 5000 g per plate. The h i ghest concentration and range of concentrations of test chemical per plate were those recommended by Mortelmans and Zeiger ( 2000) Chemicals a nd Microbiological Media Tryptic s oy agar (TS A ) was purchased from Bacto (Becton, Dickinson and Company, Sparks, MD U.S.A. ). Glucose was purchased from Difco (Houston, TX U.S.A. ) Agar powder was purchased from Fisher Scientific (Pittsburgh, PA, U S A ) Sodium azide, 9 aminoacridine and 2 Aminoanthracene were purch ased from MP Biomedicals, LLC (Solon, OH U.S.A. ). Daunomycin, top a gar supplemented with 0.6% L Histidine and D biotin, Vogel Bonner E salts (VB salts 50x), post mitochondrial supernatant (S 9), rat (Aprague Dawley ) liver, Aroclor 1254 induced, and Molto x NADPH generator (Regensys A and Regensys B) were purchased from Molecular

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60 Toxicology (Boone, NC U.S.A. ). Dulbecco s odium phosphate buffer, 0.1 mM, pH 7.4 was purchased from Sigma (Saint Louis, MO, U S .A. ). Bacteria Strains For Ames mutagenicity assay, cultures of Salmonella Typhimurium ( TA 98 and TA 100 ) were purchased from Molecular Toxicology, Inc. (Boone, NC U.S.A. ). For the antimicrobial activity assay by disc diffusion method, cultures of Alcaligenes faecali ATCC 8750, Pseudomonas putida ATCC 126 33, and Bacillus coagulans ATCC 7050 were purchased from the American Type Culture Collection ( ATCC ; Manassas, VA U.S.A. ) Ames Mutagenicity Assay The mutagenicity was evaluated on two strains of Salmonella Typhimurium (TA 98 and TA 100). The assay was p erformed as described by Mortelmans (2000) with some modifications. Glucose minimal agar plates (GM agar plates) were prepared by aseptically adding 50 mL of sterile glucose solution (10% w/v), 20 mL sterile VB salt solution and 930 mL of sterile agar at 65 C, and then mixing well with a magnetic stirrer. It was aseptically poured (25 mL of the agar medium) into each of 100x15 mm petri dishes (Fisher Scientific, Pittsburgh, PA, U S A ) The bacterial cultures were grown by picking 5 colonies of the bact eria from TSA inoculating in 25 mL of TSB broth in a flask, and then shaking 12 16 hours at 110 rpm in a water bath at 37 C (Gyrotory Water Bath Shaker, Model G76, New Brunswick Scientific Co. INC. ; Enfield, CT, U.S.A. ). The desired density of overnigh t culture was 1 2 x 10 9 colon y forming unit (CFU)/mL at an absorbance of 1.2 1.4 at 660 nm.

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61 After autoclaving, the top agar (2 mL) with 0.6% histidine and biotin were transferred to a sterile glass tube (100 x 13 mm) and kept at 48 C in a water bath unt il use. For the samples with a metabolic activation system, the S9 mix was added to NADPH generating solution (Regensys A and Regensys B). The system was prepared by rehydrating the S9 with 2.1 mL ice cold sterile deionized water and mixing well. Then 1. 6 mL of rehydrated S9 was added to the ice cold Regensys A bottle, mixed well and kept on ice until used. When ready to use, the Regensys B (NADP) was added to the Regensys A bottle and mixed thoroughly. The Ames assay was performed by aseptically pipetti ng 0.50 mL of 0.1 mM sodium phosphate buffer pH 7.4, 0.1 mL of sappanwood extract s, and 0.10 mL of overnight S almonella culture (about 1 2x10 8 cfu /mL ) into top agar (48 C), and then mixing well with a vortex. The mixture was immediately poured onto the s urface of GM agar plates and swirled quickly for even distribution of the top agar mixture. Once the top agar was solidified plates were inverted and incubated at 37 C for 48 hours. For samples where metabolic activation was required, the phosphate buf fer was replaced by the S9 mix (same volume of 0.1 mL). Negative (sterile deionized water) and positive (daunomycin) controls were included in every assay in the same manner as the test sample (0.1 mL per plate). Sterile deionized water was used as a nega tive control for all experiments. Daunomycin (60 g/mL) was use d as positive control for TA 98 assay, while for TA 100, the positive control was sodium azide (50 g/mL). The experiments were done in triplicate for each concentration, and for positive and negative controls.

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62 Results were recorded by counting the number of colonies after 48 hours of incubation, and the background of each sample dish was also compared to the negative control in the absence of the background ( thinning ) Antimicrobial Activity by Standard Agar Disc Diffusion Method The standard agar disc diffusion method was performed as described in the M anual of C linical M icrobiology (Baron and Murray 2003) Nutrient agar plates were aseptically prepared as directed by the manufacturer (23 g nutrient agar per 1 L of deionized water). Each bacterial culture was prepared by inoculating 5 colonies of the bacteria into a glass tube containing 7 mL of steril iz e d TSB. The bacterial suspensions were incubated at 37 C until the cell density of 1x10 8 cfu/mL was reached. The density was det ermined by turbidity according to McFarland standard (absorbance of approximately 0.1 at 600 nm). Freeze dried cold wat er extracts of sappanwood were reconstituted with sterile deionized water to achieve the concentration s of 0.5, 5, and 50 mg/mL. Then 30 L of the ext racts at each concentration was impregnated onto a sterile blank disc (6 mm diameter) and dried aseptically. Then the discs were aseptically placed on the surface of the nutrient agar plate, one disc each for the three concentrations was plac ed on the same petri dish along with the negative control (sterile deionized water). The experiment was repeated in triplicate. The plates were incubated for 24 hours at 37 C except for those inoculated with A. faecalis which were incubated for 48 hour s. The antimicrobial property was determined by measuring the size of inhibition zone (area where no growth of bacteria was observed) in millimeters. The experiments were done in triplicate. Extracts with inhibition zones of higher than 6.0 mm are consi dered effective.

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63 Statistical Analysis The n umber of revertant colonies data from the antimutagenicity assay inhibition zone data and the antimicrobial activity test were reported as mean s and standard s deviation. Zone inhibition data was analyzed by an analysis of variance (ANOVA) and to evaluate the difference in inhibition zone between the type and concentration s of SPW extracts. SAS 9.2 statistical software was use d (SAS Institute Inc., Cary, NC, U.S.A.). An example of the SAS output is shown in Appendix B. Results and Discussion Ames Mutagenicity Assay A m utagenicity assessment was performed on 2 types of sappanwood extracts, i.e. reflux with water for 5 hours (RF 5) and shaking for 6 hours at room temperature (RT). The mutagenicity tested on S. Typhimurium TA 98 and TA 100 of the extracts as determined by number of revertant colonies grown on the glucose minimum agar (GM) plate with histidine limited top agar is s hown in Tables 4.1 and 4.2 for RF5 and RT respectively. The RF5 was tested at five different concentrations ranging from 50 to 5000 g per plate and the number of revertant colonies of all 5 con centrations is shown in Table 4 1. The RF5 results were not higher than that of the sterile deionized water (negative control), and many folds lower t han the positive control. Because numbers of revertant colonies were not at least 2 fold higher than that of the negative control, it can be concluded tha t the RF5 d id not have any mutagenicity. RF5 with the metabolic activation system (samples with S9 fraction of rat liver added) also showed similar

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64 results of low revertants. This result suggested that, similar to the original compounds, metabolites of RF5 also ha d no mutagenicity. Table 4 2 shows the number of revertants for S. Typhimurium TA 98 and TA 100 in the presence of RT, both with and without metabolic activation system mutagenicity. Similar to results for RF5, RT results also suggest that cold water ext racts of sappanwood do not contribute to mutagenicity. According to Ames assay principles as published by Maron and Ames (1983) and by Mortelmans and Zeiger (2000) repeatable re sults from at least 2 independent laboratories are needed in order to confirm that the extracts are non mutagenic as determined by S Thyphimurium TA 98 and TA 100. Also, as a tier ed approach wa s used, further tests on different strains of S. Typhimurium are recommended. The absence of mutagenicity of sappanwood water extract from the current study agrees with the absence of toxicity of sappanwood extract evaluated in rats as well as the lack of cytotoxicity against mammalian cells (Xu and Lee 2004; Sireeratawong and others 2010) The cytotoxicity was conducted on Vero cells with brazilin concentrations ranging from 100 to 1000 g/mL. When compared with the control (no brazilin), the number of viable cells were not different (Xu and Lee 2004) In the toxicity on rats, the hot water extraction of sappanwood from Thailand was administered to five Wistar rats per gender at a dose of 5,000 mg/kg body weight. The a cute toxicity wa s determined by general behavior, mortality and changes in gross appearance of internal organs during the15 day experiment. The subacute toxicity was studied for 30 days by measuring body weight, organ weight, hematological, and blood chemicals. The aut hors concluded that

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65 the sappanwood extract did not exhibit either acute or subacute toxicity in rats (Sireeratawong and others 2010) Results from these two previous studies on the absence of toxicity for sappa nwood extract and brazilin support the data generated in this study on the absence of mutagenicity for sappanwood extract. These results also serve as scientific evidence that sappanwood extract and brazilin are not toxic to humans. In addition, the fina l concentration in food products of the sappanwood extract is self limiting because at higher concentrations the color of the product will be too intense for consumption. According to the Office of Food Additive Safety, other recommended toxicological test s include, but are not limited to, subchronic toxicity with rodents and non rodents and one year toxicity studies with non rodents. Human studies including epidemiology studies may be needed if the levels considered in foods is thought to be high for cert ain substances (C enter for Food Safety and Applied Nutrition/ U.S. Department of Health and Human Services 2006) Nevertheless, the current results support the data presented from other rel evant toxicity studies that the sappanwood extracts are neither toxic nor mutagenic. Antimicrobial Activity Evaluated by Disc Diffusion Method Antimicrobial activity using the disc diffusion method enables the comparison of antimicrobial activity among sa ppanwood extracts toward the tested bacterial strains. After reconstitution in deionized water, concentrations of the extracts were 0.5, 5.0 and 50.0 mg / mL (of freeze dried sappanwood extract); the absolute weight concentrations after the impregnation wer e 15, 150, and 1500 g per disc, respectively. Samples exhibiting clear zones of inhibition beyond the perimeter of the discs (6 mm) were

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66 determined as having antimicrobial activity (or effective) against the test bacterial strains, thus the diameters of the inhibition zones can be measured in mm. From the results (Table 4 3), a t 15 g per disc, no antimicrobial activity was observed in any sa ppanwood extracts against the test bacteria. Bacillus coagulan was the most sensitive bacterial strain to the sapp anwood water extracts (RF5 and RT) At 150 g / discs the antimicrobial activity was detected only on Bacillus coagulan with inhibition zones of 18 and 19 mm for RF5 and RT respectively. Pseudomonas putida was the least responsive to the antimicrobial act ivity of sappanwood extracts even at 1500 g per disc, the inhibition zone was about 9 mm, which is smaller compared to t hat of Alcaligenes faecalis and B. coagulans which had an inhibition zone of 24 26 mm and 33 mm respectively No significant diff erence was found for the same amount of extract per disc for the same bacterial strain when comparing the hot and cold water extraction (RF5 and RT). This indicates the possibility that both hot water extraction (RF5) and cold water extraction (RT) may ha ve nearly the same type and quantity of antimicrobial compounds. It is possible that excess heat used in the 5 hours reflux extract did not contribute to the extractable amount of antimicrobial compounds. Results obtained and discussed from this study as described above suggest the potential of sappanwood water extract as a food additive, and similar results were found from previous studies (Kim and others 2004; Xu and Lee 2004; Lim and others 2007; Phattayakorn and Wanchaitanawong 2009; Saraya and others 2009) Although results from the current study as well as others demonstrated the antimicrobial activity of the sappanwood ex tract against various types of bacteria, the mechanism s or mode of

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67 reaction are not fully understood. Further identification, isolation and quantification of active compounds responsible for antimicrobial activity, and understanding their mode of action is needed. This type of information will be beneficial to the development of sappanwood extract into a food preservative (additive), or food colorant with antimicrobial activity. Since each food system has unique properties, application of the aqueous sap panwood extract on a specific model is needed to determine the actual shelf life of each food product. Conclusion Sappanwood water extracts (RF5 and RT) did not exhibit mutagenicity as evaluated by Ames mutagenicity assay. Moreover, the extracts exhibit ed some antimicrobial activity against the growth of selected spoilage bacteria (A. faecalis, B. coagulans, and P. putida). Based on these results, sappanwood water extracts may have potential to be applied into food products both as a red colorant and as a food preservative.

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68 Figure 4 1. 5 hydroxy 1,4 naphthoquinone

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69 Table 4 1. Mutagenic dose response of RF5 sappanwood water extract to Salmonella Typhimurium (TA98 and TA100) as represented by the mean number of revertant colonies (CFU/plate) +/ sta ndard deviation (n=3) Dose level (g/plate) Number of revertant colonies (CFU/plate) TA 98 TA 100 Without metabolic activation Sterile deionized water (NC) 27.0 5.0 11.0 5.0 Daunomycin (60g/plate ; PC ) 316.7 25.4 Sodium azide (50g/pl ate ; PC ) 1118.7 200.2 5000 0.0 0.0 0.0 0.0 3333 3.3 2.1 4.7 5.0 2500 18.7 8.0 2.7 0.6 500 22.3 2.5 7.7 4.9 50 24.0 6.9 12.0 3.5 With metabolic activation (S 9) Sterile deionized water (NC) 39.0 8.0 12.0 1. 0 Daunomycin (60g/plate ; PC ) 1980.0 404.2 Sodium azide (50g/plate ; PC ) 133.7 13.6 5000 10.3 3.5 1.7 1.2 3333 16.3 7.6 6.7 1.2 2500 17.0 4.4 5.7 1.5 500 28.0 8.2 11.0 5.0 50 27.0 1.0 14.0 1.0 Values expressed are me an SD of three experiments. Two fold or more of number of revertant colonies is an indicator of mutagenicity ; NC = negative control; PC = positive control.

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70 Table 4 2. Mutagenic dose response of RT sappanwood water extract to Salmonella Typhimurium (TA9 8 and TA100) as represented by the mean number of revertant colonies +/ standard deviation (n=3) Dose level (g/plate) Number of revertant colonies (CFU/plate) TA 98 TA 100 Without metabolic activation Sterile deionized water (NC) 26.0 4.0 8.0 4.0 Daunomycin (60g/plate ; PC ) 315.0 27.1 Sodium azide (50g/plate ; PC ) 1205.3 237.5 5000 2.7 3.1 2.0 1.7 3333 12.0 2.6 6.3 3.5 2500 22.7 4.9 6.3 2.1 500 19.3 0.6 10.7 2.5 50 27.3 6.1 10.7 3.8 With metabo lic activatio n (S 9) Sterile deionized water (NC) 33.0 4.0 18.0 9.0 Daunomycin (60g/plate ; PC ) 1556.0 256.1 Sodium azide (50g/plate ; PC ) 195.0 45.3 5000 15.7 2.5 5.7 1.2 3333 19.7 7.8 9.3 4.0 2500 21.0 1.0 10.3 3.8 500 33.0 2.6 8.3 2.9 50 31.7 2.3 12.0 2.0 Values expressed are mean SD of three experiments. Two fold or more of number of revertant colonies is an indicator of mutagenicity; NC = negative control; PC = positive control.

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71 Table 4 3. Antiba cterial activities of sappanwood water extracts against three strains of food spoilage bacteria. Wt. of freezed dried Inhibition zone (mm) RF5 RT A. faecalis Sterile deionized water 0.00 0.00e 0.00 0.00e 1500 A26.33 0.58b A24.33 1.53b 150 0.00 0.00e 0.00 0.00e 15 0.00 0.00e 0.00 0.00e B. coagul ans Sterile deionized water 0.00 0.00e 0.00 0.00e 1500 A33.33 0.58a A32.66 0.58a 150 A19.33 1.15c A18 1.73c 15 0.00 0.00e 0.00 0.00e P. putida Sterile deionized water 0.00 0.00e 0.00 0.00e 1500 A8.67 1.15d A 8.67 0.58d 150 0.00 0.00e 0.00 0.00e 15 0.00 0.00e 0.00 0.00e Values expressed are mean SD of three experiments. Extracts with inhibition zone higher than 6.0 mm are considered effective. Different upper case letters in front of inhibition zone values in the same row s and different lower case letters in column s represent value s that are significantly different ( p < 0.5).

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72 CHAPTER 5 STABILITY OF RED COL OR FROM SAPPANWOOD C OLD WATER EXTRACT IN AQUEOUS SOLUTION S Background Overview Ideal prope rties of food color additives include stability in a wide range of colorant concentrations, the pH of products heat treatment sulfur dioxide exposure and storage conditions (such as temperature and exposure to air and light) that are common in processed food products. The c olor of the finished products should be vibrant with no off flavor The raw materials should be inexpensive, free of insec ticides, herbicides or microbial contamination, and they should not be seasonal dependent. The y ield of the ex traction should b e high and the colorant s obtained should have high color strength. Moreover, raw materials of plant origins may be preferred as they can be qualified as vegetarian, and kosher approved ( Downham and Collins 2000 ; Kilcast and Subramaniam 2000; Wrolstad 2004; Griffiths 2005; Castaeda Ovando and others 2009 ; Nachay 2009) The s tability over a wide range of concentration needs to be evaluated because it can be unpredictable. Normally, a s concentration increases, colorant solutions will show higher color saturation (higher chroma value), while the color shade (hue) is less affected by concentration Exceptions are, f or example, cyanidin solution which is red at high concentration s but change s to purple hue s at low concentration s (Hutchings 1999) Susceptibility to degradation unde r heat and light exposure is a common character istic of plant derived colorant s such as anthocyanins, w hich are avai lable commercially as red colorant s from various plant materials Another important factor that causes instability of color is pH. Most anthocyanins, for example, change color and

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73 become instable at pH levels above 4 These factors are impo rtant as they are major limitations to the a pplication of colorant s in food (Wrolstad 2004) The molecular structure of brazilin, a compound responsible for the red color of sappanwood cold water extract, is considered a homoisoflavonoid, which is a su bgroup of flavonoid. Brazilin is a reduced form and it is the form that naturally exists in sappan wood. W hen expose d to oxygen and light, it is oxidized to brazilein, thereby changing color from colorless (brazilin) to red (brazilein) (Vankar 2000; Ferreira and others 2004; Wongsooksin and others 2008; Berger and Sicker 2009; Petroviciu and others 2010) It should be noted that prior to this current study, there was no study on the stability of braziliein or sa ppanwood extract. The objective of the present study is to investigate the stability of aqueous sappanwood extract as affected by concentration, heat and light. The results obtained can be useful in furthering the development of sappanwood extract as a c olor additive in food products. Materials and Methods Raw Materials and Chemicals Sappanwood was purchased from Jao Krom Poe the oldest and most reputable Thai herb pharmacy in Bangkok, Thailand (Usuparatana 1997) Potassium chloride, hydrochloric acid, citric acid, dibasic sodium phosphate, dibasic hydrogen phosphate, monobasic sodium phosphate, and glycine were purchased from Fisher Scientific Co., (Pittsburgh, PA, U S A ). The p reparation of pH 7, 8 and 9 buffers was completed as described in Table 3 1.

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74 Sappanwood Extracts Preparation Preparation of the extract was described in Chapter 3. The f reeze dried s appanwood water extract (RT) was used to evaluate concentration effects However, for heat stabili ty and storage stability evaluations the cold water extracts were used without further freeze drying step. After th e extraction and filtration as described in Chapter 3, extracts were blanched by heating the extract s to 212 F for 10 minutes, and then co oled down immediately on ice water ( Cevallos Casals and Cisneros Zevallos 2004) Th e concentration of the extract was standardized when the temperature of the extract reached 25 C by diluting it with deionized water until an absorban ce of 1.0 at 539 nm wa s reached for the solution of the extract at pH 9 (extract : buffer pH 9 = 1:6 ) T he absorbance was obtai ned by a spectrophotometer (Beckman Coulter DU 640; Indianapolis, IN, U S A ). The standardized extracts were kept refrigerated (4 C) then used within 24 hours after preparation. Color M easurements Color characteristics as recommen ded by The International Commission on Illumination (CIE) were measured as L*, a*, b*, hue, and chroma using a Minolta CT 310 colorimeter (Minolta Corporation, Ramsey, NJ U.S.A. ). The solution of the extract and buffer was placed in a 10 mm plastic cell ( Minolta CM A131), and put in the Minolta cell holder (CR 400). The colorimeter was set up using the light source D65. The CIE L*, a*, b*, hue, and chroma values of each sample obtained were average s of the duplicate measurements and were read directly f rom the colorimeter. The change of color was quantified as the metric distance between two colors, represented as the

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75 re flectance data: L*, a*, b* used the following formula, where L 0 a 0 and b 0 are the reference values at time 0 (Medeni 2001) Spectrophotometric A nalysis The absorbance of solutions of sappanwood extract in buffer from the wavelength of 250 to 700 nm was measured by a UV vis spectrophotometer (Beckman Coulter DU 640; Indianapolis, IN, U S A ) using disposable cuvettes (1 .5 mL purchased from Fisher Scientific, Pittsburgh, PA, U S A ). Percent color retention at max was calculated using the following formula, where Abs 0 is the absorbance at time 0. % color retention at max = [(Abs Abs 0 ) / Abs 0 ] x 100 Color Stability Study o f Sappanwood Extract s Under Different Conditions Thermostability The r mostability (at 80 C ) of the sappanwood water extract (at pH 7, 9, and 12 ) was evaluated under three different conditions ( light and air ; no light and air; no light and no air ) The temperature control ( 80 82 C ) was accomplished by submerging sample tubes (glass) in a water bath shaker (Gyrotory Model G76, New Brunswick Scientific Co., INC. ; Enfield, CT, U.S.A ). In each glass tube (16 x 100 mm), 2.0 mL of standardized sappanwood extract was added to 12.0 mL of buffer pH 7, 9, and 12. Sample tubes for each treatment condition were prepared in duplicate. After tightly closing with a screw cap and mixing well with a vortex, all tubes were then immediately placed in the water bath shaker. Samples were pul led out at 0, 0.5, 1.0, 2.0, 3. 0, 5.0 and 7.0 hours, cooled immed iately in ice water, and then analyzed for color and UV vis

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76 spectra. No air condition is defined as flushed with nitrogen and no light means it was performed in the dark. Effects of c oncentration s on c hromaticity The freeze dried powder from the cold wat er extraction of sappanwood (RT) as described in chapter 3 was dissol ved in warm deionized water (approximately 60 C) t o achieve concentrations of 5, 10, 20, 40, 50, 1 00, and 200 g/mL. The dilution scheme was described in Appendix C. Each concentration was prepared in triplicate (3 test tubes per concentration). All samples were analyzed for color measurements. Storage s tability To evaluate the stability of red color from sappanwood extract during storage, sappanwood extract at the same concentration s ( pH 7, 8, and 9) were stored under 3 different conditions (room temperature with light; room temperature no light ; and at 4 C, no light ). In each glass tube (16 x 100 mm), 2.0 mL of standardized sappanwood extract was ad ded to 12.0 mL of buffer s pH 7, 8 and 9 Sample tubes for each treatme nt condition were prepared in tri plicate (54 tubes were prepared for each pH) After tightly closing with a screw cap and mixing well with a vortex, samples tubes were placed in the 3 different conditions Samples w ere pul led out at days 0, 3, 7, 14, 21, and 35, then analyzed for color and UV vis spectra. Light condition was defined as 24 hour exposure to fluorescent light per day, and no light means it was performed in the dark. Buffer pH 7, 8, and 9 was prepared as described in Chapter 3. Screw cap test tubes (16 x 100 mm) containing 12 mL buffer were autoclaved at 121 C for 30 minutes then kept at room temperature until ready to use.

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77 Statistical Analysis Redness (a* value) % color retention, chroma, hue angl e and color difference data were reported as mean and standard deviation. Color difference data was analyzed by analysis of variance (ANOVA) and mean separations were performed by studentized range test (p < 0.05) in order to evaluate the differe nce in the color difference between storage time and storage conditions. SAS 9.2 statistical software was used (SAS Institute Inc., Cary, NC, U.S.A.). An example of the SAS output is shown in Appendix B. Results and Discussion Thermostability The thermal stability ( 80 C ) of the extracts was evaluated by exposing the sappanwood extracts ( pH 7, 9 and 12 ) for a total of 7 hours in different experi mental condition s (light and air; no light and air; no light and no air). As sappanwood extract is a product of plant origin, blanching of the extract was performed to deactivate any polyphenoloxidase enzymes that may be presented as described by Cevallos Casals and Cisneros Zevallos (2004) for 100% deactivation of the enzymes in other plant materials. The effects of heat exposure at di fferent pH levels was evaluate d by the CIE color system ( a*, hue, and chroma ) and spectrophotometric analysis expressed as % color retention at max (539 nm) Figures 5 1 showed changes in a* value (redness) during heating of the sappanwood extracts The redness for all extracts (pH 7, 9, and 12) was reduced wi th longer heating duration. However, redness of sappanwood solution at pH 12 was reduced to less than 0.00 after only 0.5 hour s of heating time while solution s at

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78 pH 7 and 9 were higher than 15.00 after 2 hours of heating. This result indicates that the heat stability of sappanwood ex tract at pH 12 is very low. Similar to the previous report in chapter 3 UV vis spectra of the sappanwood extracts at the various pH values showed maximum absorption in the visible region at wavelength of 539 nm ( max = 539 nm). Comparison of UV vis spectra of sappanwood extracts at pH 9 showed that the absorbance at 539 nm was reduced with the duration of h eating (Figure 5 2 ). A c omparison of the color retention at 539 nm of sappanwood extr act pH 9 was shown in Figure 5 3 with best color retention demonstrated when samples were heated without exposure to light and air (approximately 70% after 7 hours of heating at 80 C) When air is present the % color retention at 539 nm was reduced to less than 20% regardle ss of light exposure. Sappanwood extract at pH 12 was excluded in the subsequent experiment as its thermal stability is very low. In addition, the pH of food is unlikely to be higher than 9. Thus, pH of 7, 8, and 9 were selected for further stabilit y studies. Concentration Effects o n Color o f Sappanwood Extracts Aqueous solutions of sappanwood extract at pH 7, 8, and 9 were pre pared at concentrations of 5 2 00 g/mL. Saturation of color or color intensity is represen ted by chroma, which was calcula ted as (a* 2 + b* 2 ) 1/2 As illustrated in Figure 5 4 intensity of color in term of chroma value s of sappanwood extracts at pH 8 and 9 was increased as the concentration of the freeze dried sappanwood in the solution increased from 5 40 L/mL, and then was reduced For SPW extract at pH 7, the chroma values continued to increase with the increase of the concentration to the concentration of 200 L/mL, which was the highest concentration tested. For colorant s it is desirable to have co lor

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79 intensity increase with the concentration of color Thus, this experiment showed that sappanwood extracts met this required quality up to a chroma value of approximately 34 36 for pH 8 and 9, and up to a chroma value of 40 for pH 7. The next import ant param eter is the visual color itself; thus actual photo s for visual color comparison are shown in Figure 5 5 The visual colors agreed with the chroma values measured, i.e. colors were in the same shade that is light orange to dark orange in pH 7, a nd light pinkish red to intense red for pH 8 and 9. C olor saturation was higher as concentration s increased until the maximum was reached for pH 8 and 9 (chroma values of 34 36), while color saturation of extract at pH 7 was highest at the highest con centration (200 g/mL) Hue angle was be calculated as arctan b*/a* and expressed as degree on a 360 grid. Close to 0 is bluish red, and as the value approaches 90 a more yellowish to yellow color results Hue angle should remain the same while chroma increases as concentration of colorant increases (Wrolstad and others 2005) The hue angle of all samples increased with the concentration of sappanwood extract in the buffer solution (Table 5 1 ). The range of hue angle suggested that the direction of bluish red toward orange red as colorant concentration increased for the pH 9 solution. Solutions at a pH 9 also had the most stability (least degree of change) toward concentration changes because, of all 7 concentration s tested, there were only 4 groups of mean hue angle s th at were significantly different p < 0.0 5) followed by a total of 5 groups of mean s in pH 7, and 7 groups of mean s in pH 8

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80 Storage Stability Visual colors of sappanwood water extra ct solutions at pH 7, 8, and 9 were shown in Figure 5 6 For comparison in absolute values, c determine the effect of pH, storage temperature, and light exposure during storage of the sappanwood extract (Table 5 2) Storage at room temperature, both with and without ligh t significantly increase d color difference ( E ) for all sappanwood e xtracts Storage at 4 C in the dark contribute to significantly less color difference for all extracts after 35 days of storage for all pH levels (7, 8, and 9). The only e xception is for pH 9 which, after 35 days showed no signifi cantly difference in E was observed between storage at room temperature and storage at refrigerator temperature when stored in the dark Thus, eliminating light exposure, even when store d at room temperature, can significantly prolong the color of sappan wood extract at pH 9. For the extract at pH 7 and 8, storage at refrigerator temperature without light exposure had significantly less E than the other two conditions (room temperature with and without light exposure). From these results, sappanwood sol ution at pH 9 had better storage stability when compared to the sappanwood solution s at pH 7 and 8. When compar ing the E (color difference) of sappanwood extract at pH 9 over storage time in different experimental condition s it can be observed that the r e was no significant difference in E between Day 0 and Day 14. There was also no significant difference between E on day 14 of all 3 experimental conditions. This could be an indication that, at a pH of 9, the sappanwood solution can be stored in eith er room or

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81 refrigerator temperature, either with or without light exposure for up to 14 days with no significant changes in color of solution based on color difference values Conclusion Stability of sappanwood extracts at various pH values was tested. The solution of sappanwood extract at pH 9 showed the highest stability to heat, light and oxygen. A higher concentration did not dramatically change the shade of color, which is a desirable property for a color additive. Results obtained from this st udy suggest the factors that contribute to the higher stability of red color are controlling the pH to pH 9, limiting light and oxygen exposure, and low temperature storage. Further investigation on the degradation mechanisms of brazilein and how to retar d these reactions could lead to sappanwood as a potential color food additive.

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82 Figure 5 1 Effects of pH ( 2, 5, 7, 9, and 12 ) on heat stability of sappanwood extracts in term s of a* value (redness) with light and air exposure Figure 5 2 UV vi s spectra of sappanwood extract at pH 9 duri ng heating (80 C) with light and air exposure. -5.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 more yellow < -a* -> more red Time (hour) pH 7 pH 9 pH 12 0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 0.6000 0.7000 0.8000 0.9000 1.0000 400.0 450.0 500.0 550.0 600.0 650.0 700.0 Absorbance Wavelength (nm) 0.0 h 0.5 h 1.0 h 2.0 h 3.0 h 5.0 h 7.0 h

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83 Figure 5 3 Effects of heat on % color retention of sappanwood extracts (at 539 nm) at pH 9 under different environments. The b lue line represents light and a ir exposure; the red line represents air exposure but no light; and the green line represents no heat and no air exposure. Figure 5 4 Effect s of sappanwood extract concentrations on color saturation (chroma) at pH 7, 8 and 9. 0.0000 20.0000 40.0000 60.0000 80.0000 100.0000 120.0000 0.0 2.0 4.0 6.0 8.0 %Color retention at Abs. max (539 nm) Time (hour) light and oxygen exposure dark with oxygen exposure dark with no oxygen exposure 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 Chroma Concentration (mg/mL) pH 7 pH8 pH9

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84 Figure 5 5 Visual colors of different sappanwood extract concentrations at pH 7, 8 and 9 (high to low from left to right). Table 5 1. Effects of various concentrations of sappanwood water extract s on hue angle at pH 7, 8, and 9 Concentration ( g/mL) Hue angle pH 7 pH 8 pH 9 200.0 45.83 a 0.51 389.94 a 1.07 377.63 cd 0.84 100.0 47.05 a 0.09 376.58 b 1.42 360.10 cd 0.54 50.0 39.15 a 0.22 360.81 c 1.90 344.75 cd 0.10 40.0 34.61 c 0.47 354.97 d 0.26 341.76 c 0.24 20.0 23.05 d 0.39 347.84 e 0.28 342.71 c 11.48 10.0 11.92 e 1.03 344.89 f 0.23 333.94 b 0.30 5.0 358.27 f 1.17 341.95 g 0.17 332.26 a 0.34

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85 Mean v alues (n=3) followed by different lowercase letters in the same column are 0. 0 5). pH 7 pH 8 pH 9 Figure 5 6 Sappanwood water extract after 35 days in different environments, from left to ri ght of each picture, at 25 C with light exposure at 25 C in the dark, and at 4 C in the dark

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86 Table 5 2. Color difference s during storage of sappanwood extract s under various storage conditions and pH Condition Color Difference ( ) 0 day 3 day 7 day 14 day 21 day 35 day pH 7 25C, Light A 0.00 0.00 f B 3.11 0.72 e A 17.99 0.54 d A 23.79 0.08 c A 25.13 0.32 b A 50.60 0.04 a 25C, Dark A 0.00 0.00 e B 2.17 0.07 e B 9.78 0.50 d B 19.42 2.78 c A 23.41 1.03 b A 49.37 0.57 a 4C, Dark A 0.00 0.00 e A 11.27 0.13 d C 4.76 0.23 d C 5.24 0.57 c B 9.23 0.83 b B 36.69 0.76 a pH 8 25C, Light A 0.00 0.00 e A 5.56 1.80 d A 21.10 2.02 c A 36.54 0.57 b A 38.14 0.16 b A 42.95 0.16 a 25C, Dark A 0.00 0.00 c B 2.45 0.21 c B 5.49 0.5 2 c A 17.52 1.41 b B 33.01 1.56 ab A 41.22 1.23 a 4C, Dark A 0.00 0.00 d B 2.62 0.16 cd C 1.00 0.41 bc B 2.64 0.20 bc C 3.55 1.04 b B 10.48 1.36 a pH 9 25C, Light A 0.00 0.00 c A 2.06 0.02 c B 2.36 0.46 c A 3.02 0.94 c A 29.16 3.89 b A 43.72 0.13 a 25C, Dark A 0.00 0.00 b AB 3.18 0.57 b A 4.02 0.41 b A 1.96 0.92 b AB 4.22 2.17 b AB 10.19 3.66 a 4C, Dark A 0.00 0.00 c B 1.20 0.60 bc B 2.65 0.35 bc A 1.62 0.91 b B 2.13 0.16 b B 4.81 0.94 a Values expressed are mean s SD ( n = 3 ) Different upper case let ters in front of E values in the same column and differ ent lower case letters in row of each pH represent value that are significantly different ( p < 0.5).

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87 CHAPTER 6 SUMMARY AND CONCLUSI ONS The current research explored the possibility of developing a natural derived red colorant as a food additive from sappanwood water extract B ased on the current lit erat ure, th e research focused on the water extracts and i t was f ound that water extraction at room temperature (RT) and reflux at 5 hours (RF5) provided the better color saturation and redness, compared to the longer reflux time (RF24). This study is the first to report the chromaticity of the sappanwood water extract at pH 2 12 The results suggested the potential advantage of using the sappanwood water extract as a naturally derived red colorant for pink to red color s at a higher pH, which has been a limitation of the anthocyanins in food application. Based on the Ames mutagenicity test, water extracts ( RT and RF5 ) were not mutagen ic Cold water extraction (RT), thus, was chosen for further evaluation because it required less energy during extraction. RT water extract was sensitive to heat, light and temperature and t he best storage condition was at pH 9 and 4 C (refrigerator) with no light exposure. In a ddition sappanwood extract may be beneficial to health as indicated by antioxidant activities reported The r elatively low antimicrobial activity reported he r e suggested little potential for using sappanwood extract as a food preservative when used at concentration s for coloring purpose s Overall, this research provided evidence for further exploration of sappanwood water extract as a potential color food addi tive T he main advantage s include long history of consumption as well as an ability to give red color to low acid food s

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88 APPENDIX A EFFECTS OF EXTRACTION TIME ON MAXIMUM ABSORBANCE OF SAPPANWOOD COLD WATER EXTRACTION Background Cold water extraction may be desirable compared to hot water extraction due to fewer unwanted compounds being extracted. Thus, cold water extract is an interesting subject to study red colorant from sappanwood. T he t otal time of extraction can affect the yield, and should be det ermined as a preliminary study. Materials and Methods In an Erlenmyer flask, 3.0 g of sappanwood powder was added to 100 mL of deionized water and shaken for 2, 4, 5, 6, 7, and 8 hours on a platform shaker (New Brunswick Scientific Excella E1 Platform Shak er ; Edison, NJ, U.S.A. ). The solution obtained was first filtered through Whatman #1 filter paper and then through 0.25 M syringe filter. The UV vis spectra was collected in the extract from 400 600 nm. The experiment was done in triplicate. Res ults and Discussion The UV vis spectra of the 6 treatments (Figure A 1) have similar characteristics, i.e. having maximum absorbance at between 444 to 448 nm. The results were agreed with previous studies which indicated the absorbance max of brazilein at 445 nm (Yan and others 2007) Since the 6 hour extraction yield the highest absorbance at 445 nm, this method of extraction w a s selected for the subsequen t steps of this study.

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89 Figure A 1 Effect s of extraction t ime on UV vis spectra ( from 400 to 600 nm ) of sappanwood cold water extracts. Figure A 2 Effect s of extraction time on absorbance at 445 nm of cold water extracts of sappanwood powder. Data represents the mean of n=3 Colu mns with similar le tters 0.05) 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000 0.4500 0.5000 400 450 500 550 600 Absorbance Wavelength (nm) 2 hr 4 hr 5 hr 6 hr 7 hr 8 hr 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 2 4 5 6 7 8 Absorbance at 445 nm Extraction time (hours) a bc c bc d b

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90 APPENDIX B EXAMPLE OF S TATISTICAL ANALYSIS The ANOVA Procedure Class Level Information Class Levels Values extraction 3 RF24 RF5 RT Number of Observations Read 9 Number of Observations Used 9 Dependent Variable: GAE Sum of Source DF Squares Mean Square F Value Pr > F Model 2 418.8534000 209.4267000 13.82 0.0057 Error 6 90.8996000 15.1499333 Corrected Total 8 509.7530000 R Square Coeff Var Root MSE GAE Mean 0.821679 4.278027 3.892292 90.98333 Source DF Anova SS Mean Square F Value Pr > F extraction 2 418.8534000 209.4267000 13.82 0.0057 Tukey's Studentized Range (HSD) Test for GAE NOTE: This te st controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error De grees of Freedom 6 Error Mean Square 15.14993 Critical Value of Studentized Range 4.33920 Minimum Significant Difference 9.7511 Means with the same letter are not significantly different. Tukey Grouping Mean N extraction A 98.983 3 RF5 A B A 91.653 3 RT B B 82.313 3 RF24

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91 APPENDIX C PREPARAT ION OF SAPPANWOOD EXTRAC T SOLUTION S AT VARIOUS CONCENTRATIONS FOR C OLOR STABILITY TE ST ING Table C 1. Preparation of sappanwood extract solution s at various concentration s Working solution preparation In each test tube Solution # Wt. of RT* (g) Solution # and vol. (mL) Water (mL) Conc of working solution (mg/mL) vol (mL) of working soluti on vol (mL) of buffer Conc of final solution (mg/mL) A 0.100 0 n/a 50 2.0 00 3 12 0.400 B n/a A = 25 25 1.00 0 3 12 0.200 C n/a B = 20 20 0.50 0 3 12 0.100 E n/a B = 10 30 0.25 0 3 12 0.050 F n/a A = 6 54 0.20 0 3 12 0.040 G n/a F = 30 30 0.10 0 3 12 0.020 H n/a G = 20 20 0.050 3 12 0.010 I n/a G = 10 30 0.025 3 12 0.005 *Freeze dried sappanwood extract at room temperature as described for RT in Chapter 3.

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92 APPENDIX D CHEMICAL OXIDATION OF BRAZILIN TO BRAZILEIN Background The main colorant in s appanwood is brazilin, which in a reduced form is colorless. The color of sappanwood extract becomes red when it is changed to the oxidized form, brazilein. Molecular structures for these compounds are described earlier in Chapter 3 In this preliminar y experiment, the oxidation of brazilin as described by Wongsooksin and others (2008) was used t o explain the possible oxidation mechanisms of phenols to quinones by reagents such as iodine (Becker 1965; Barret and Daudon 1990) Brazilin and its oxidized form were separated by liquid chromatography, and a comparison of HPLC profiles help to investigate changes resulting from these oxidation mechanisms. Mass spectrophotometry has been used as to identify brazilin and bra zilein against previously published mass spectra of these compounds (Hulme and others 2005) Although the oxidation of brazilin to brazilein was mentioned in several references, none was a direct com parison (before and after oxidation) using relative new technology su ch as mass spectrophotometry. The objective of this experiment is thus to identify brazilin in sappanwood extract as well as to influen ce the oxidation of the molecular structure of braz ilin. Materials and Methods Brazilin was purchased from MP Biomedicals (Solon, OH, U S A ), HPLC grade methanol and iodine was purchased from Fisher Scientific ( Pittsburgh, PA, U S A ). The i odine solution was prepared by dissolving 33.8 g of iodine i n 42.5 mL of methanol. The b razilin solution was prepared by dissolving 100 mg of brazilin in 0.5 mL warm methanol and mixing with 8.0 mL of hot deionized water ( approximately 95 C). Once the brazilin

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93 solution was cooled to 60 70 well. The mixture was left overnight at room temperature. Brazilein, as a precipitate, was collected on filter paper (Whatman #1). The filtrate was washed several times with cold deionized water, f ollowed by warm methanol. The brazilein obtained appeared to be a deep reddish black color, it was dried in desiccator away from light, and then kept frozen in a tightly sealed glass vial. The LC/MS analysis was performed by the Chemistry Department Mass Spectrometry Services facility, University of Florida Chemistry Laboratory, by Dr. Jodie Johnson. The HPLC analysis was performed the Agilent HPLC system 1 100 series binary pump (Agilent; Palo Alto, CA U.S.A. ), equipped with Agilent 1100 G1314A UV/Vis de tector at wavelength 254 nm. Compounds were separated on Phenomenex Synergi 4u Hydro RP 80A (2 x 150 mm; 4 um; S/N=106273 5) column with C18 guard column (2mm x 4 mm) (Torrace, CA U.S.A. ). Mobile phases consisted of 0.2% acetic acid in H 2 O (phase A) and 0.2% acetic acid in methanol (phase B) and all solvents and reagents were purchased from Burdick & Jackson (Morristown, NJ, U S A ). Flow rate was 0.17 mL/min. The gradient elution started with 100% of phase A for 45 minutes, then change to 5% phase A:9 5% phase B from 45 to 60 minutes. Electrospray ionization mass spectrometry (ESI MS) was performed with ThermoFinnigan (San Jose, CA U.S.A. ), LCQ with electrospray ionization (ESI) in negative mode; and heated capillary temperature was 250 C. Injection volume was 20 L. Results and Discussion HPLC chromatograms of brazilin and oxidized brazilin samples analyzed via reverse phase gradient C18 HPLC/254 nm UV/(+) & ( ) ESI MSn are shown in Figure D

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94 1 and Figure D 2 respectively. Brazilin samples contain b oth brazilin (MW 286) and brazilein (MW 284) while the oxidized sample contains only brazilein with no brazilin. In the mass spectrometry with electrospray ionization in negative ion modes, MW 286 brazilin produced mainly the ion [M H] with m/z 285, while the MW 284 brazilein produced predominantly the ion [M H] with m/z (Figure D 3 and Figure D 4 respectively) Further dissociation of the [M H] ions of brazilin and brazilein produce product ions that agree with the previously published spectra by Hulme and others (2005) as detailed in Chapter 3.

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95 Figure D-1. Chromatogram of brazilin sample analyzed via C18 HPLC/UV/(-)ESIMS n Brazilin and brazilein ion peaks are shown in the shaded area

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96 Figure D-2. Chromatogram of oxidized brazilin sample via C18 HPLC/UV/(-)ESIMS n There are 2 brazilein ion peaks which are shown in shaded area.

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97 Figure D-3. Mass spectrum of brazilin (top) sample and oxidized brazilin sample (bottom) with (-)ESIMS

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98 Figure D-4. The [MH] ions of brazilin (top) and brazilein (bottom) were dis as sociated to produce a number of product ions.

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99 LIST OF REFERENCES Armstrong WP. 1992. Logwood and Brazilwood: Trees That Spawned 2 Nations. 53(1):38 43. Badami S Geetha B, Sharma SV, Rajan S, Suresh B. 2007. Microwave assisted rapid extraction of red dye from Caesalpinia sappan heartwood. Natural Product Research 21(12):1091 1098. Badami S Moorkoth S, Rai SR, Kannan E, Bhojraj S. 2003. Antioxidant activity of Caesalpinia sappan heartwood. Biological & Pharmaceutical Bulletin 2 6(11):1534 1537. Bae IK, Min HY, Han AR, Seo EK, Lee SK. 2005. Suppression of lipopolysaccharide induced expression of inducible nitric oxide synthase by brazilin in RAW 264.7 macrophage cells. European journal of pharmacology 513(3):237 242. Baron EJ, Mur ray PR. 2003. Manual of clinical microbiology. Washington, D.C.: ASM Press. Barret R, Daudon M. 1990. Oxidation of phenols to quinones by bis(trifluoroacetoxy)iodobenzene. Tetrahedron letters 31(34):4871 4872. Batubara I, Mitsunaga T, Ohashi H. 2009. Scre ening antiacne potency of Indonesian medicinal plants: antibacterial, lipase inhibition, and antioxidant activities. Journal of Wood Science 55(3):230 235. Batubara I, Mitsunaga T, Ohashi H. 2010 Brazilin from Caesalpinia sappan wood as an antiacne agent. The Japan Wood Re search Society 56:77 81 Becker H D. 1965. Quinone Dehydrogenation. I. The Oxidation of Monohydric Phenols. The Journal of organic chemistry 30(4):982 989. Berger S, Sicker D. 2009. Classics in spectroscopy : isolation and structure eluci dation of natural products. Weinheim: WILEY VCH. B urrows A. 2009. Palette of Our Palates: A Brief History of Food Coloring and Its Regulation. Comprehensive Reviews in Food Science and Food Safety 8(4):394 408. Bykbal ci A, El SN. 2008. Determination of I n Vitro Antidiabetic Effects, Antioxidant Activities and Phenol Contents of Some Herbal Teas. Plant foods for human nutrition 63(1):27 33. Castaeda Ovando A, Pacheco Hernndez MdL, P ez Hernndez ME, Rodrguez JA, Galn Vidal CA. 2009. Chemical studies of anthocyanins: A review. Food Chemistry 113(4):859 871.

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108 BIOGRAPHICAL SKETCH Vale eratana Kalani Sinsawasdi was born in Hawaii and raised in Bangkok, Thailand. She earned her bachelor degree in Food Science from Chian gmai University in Thailand then worked in the international marketing department of Griffith Laboratories She atte nded the University of Hawaii and earned a ma Food Science through an Asia Pacific Scholarship. A fter finishing graduate school she worked in California as a n HACCP coordinator for Stockton Further Processing also at the same time, as a HACCP consultant for Angelina Foods. Then she worked as a Quality Assurance Manager for D ean Food s in the largest rBST free milk processing plant on the West Coast until moving back to Thailand. In Bangkok, she worked for Unilever Thai Trading Ltd. H er f irst position was as a Product Development Manager for the local food d ivision Her last position before leaving the food in dustry for academia was as a Food Science Manager for Unilever Health Institute Asia which serv ed all Unilever companies in Asi a and Australia During her current position at Mahidol Uni versit y International College (MUIC), she taught many undergraduate level courses in the Food Science Department She will continue to work with MUIC after completing her Ph.D. in Food Scienc e at the University of Florida.