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UNIVERSITI PUTRA MALAYSIA
CHEOK CHOON YOONG
FK 2012 29
EXTRACTION OF TOTAL PHENOLIC AND TOTAL MONOMERIC ANTHOCYANIN CONTENTS FROM MANGOSTEEN (GARCINIA
MANGOSTANA L.) HULL
ii
EXTRACTION OF TOTAL PHENOLIC AND TOTAL MONOMERIC
ANTHOCYANIN CONTENTS FROM MANGOSTEEN (GARCINIA
MANGOSTANA L.) HULL
By
CHEOK CHOON YOONG
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in
Fulfilment of the Requirements for the Degree of Doctor of Philosophy
July 2012
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DEDICATED TO
my beloved father who passed away on 29 October 2008. I am thankful and
grateful for his sacrifices and determination to enable his children to get an
education by emphasizing the Chinese proverb “Poor in wealth but rich in
education”. My motivation in pursuance of this PhD program comes from his
determination. He always felt proud of his children’s enthusiasm in pursuing
further education degrees and acquiring new knowledge. In further appreciation of
his sacrifices, I selected mangosteen, which was cultivated by him, as a sample
source for this PhD research.
and to my sister, who passed away on 3 November 2012. She was also my
mathematic and English teacher.
In memory
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of
the requirement for the Degree of Doctor of Philosophy
EXTRACTION OF TOTAL PHENOLIC AND TOTAL MONOMERIC
ANTHOCYANIN CONTENTS FROM MANGOSTEEN (GARCINIA
MANGOSTANA L.) HULL
By
CHEOK CHOON YOONG
July 2012
Chairman : Chin Nyuk Ling, PhD
Faculty : Engineering
Mangosteen is a popular local tropical fruit because the pulp is delicious and has high
value of nutrient and mineral content. However, mangosteen hull is disposed of as waste
by local people due to the unpleasant taste of bitterness, though it contains total phenolic
and anthocyanins which have numerous important pharmaceutical properties. Due to the
remarkable relationship between total phenolic content (TPC) and total monomeric
anthocyanin (TMA) with antioxidant capacity, a series of investigations were undertaken
to enhance the mangosteen crude extract yields evaluated in terms of TPC and TMA.
This research involved investigations from fundamental to optimization, based on
hypotheses of methanol, acidic condition of extraction solvent, and the ultrasonic wave
treatment could give higher extraction yield. Results were expressed in mg gallic acid/g
hull powder for TPC, while for TMA, expressed in mg cy-3-glu/g hull powder.
The effect of solvents, i.e. ethyl acetate, isopropanol, acetone, ethanol, methanol, and
distilled water were evaluated on the extraction of total phenolic content (TPC) from
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mangosteen. Methanol was found as the most effective solvent in giving the highest
recovery (p < 0.05) of TPC, compared to ethanol, acetone, isopropanol, ethyl acetate and
distilled water. Regression equations of strong coefficients of determinations
(R2 0.990) were obtained for UV-Vis spectrophotometer direct absorbance of acetone,
ethanol, and methanol extracts with the Folin-Ciocalteu (FC) method, suggesting
regression equations obtained can be used to determine TPC without using the FC
method.
The optimum conditions of extraction time, solid to solvent ratio, and methanol
concentration for extraction of total phenolics from mangosteen hull powder was
investigated using the response surface methodology (RSM). The experimental data
obtained was adequately fitted into second-order polynomial models with R2 of 0.897.
Optimal extraction parameters which gave a maximum TPC yield of 140.66 mg (gallic
acid equivalent) GAE/g powder were from a 2 hours‘ extraction with 0.05 solid to
solvent ratio and at 69.77% methanol concentration. Analysis using artificial neural
network (ANN) predicted data which showed a higher R2 value of 0.945 and average
absolute deviation values of 4.01% versus 5.37% for the RSM. This suggests that ANN
is a better modeling technique for nonlinear data for predicting TPC yield extracted from
mangosteen hull powder compared to the RSM.
The recovery of anthocyanins from mangosteen hull was investigated using aqueous
methanol solvent acidified by juice from Mexican lime (C. aurantifolia Swingle) in
comparison with the conventional HCl acidified methanol solvent. The addition of
0.20 ml lime juice/ml methanol aqueous extraction solvent gave a maximum total
monomeric anthocyanin (TMA) recovery of 4.742±0.590 mg cy-3-glu/g hull powder
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compared to 2.950±0.265 when using HCl acidified extraction solvent at 0.20%. A
significant increase of 60.75% (p < 0.05) anthocyanins recovery suggests that this
natural Mexican lime juice can be a good acidifying agent and replaces HCl. Particle
size hull powder of 250 m to 500 m was also found to give the highest recovery of
anthocyanins (p < 0.05).
Two types of extraction solvents, i.e., methanol and ethanol aqueous solvent acidified
with 0.20% HCl were used to investigate the effect of ultrasonication time and
amplitude on TMA and TPC from mangosteen hull. The highest TMA recovery of
2.917±0.039 mg cy-3-glu/g hull powder was achieved using methanol 0.20% HCl
acidified aqueous solvent with a pretreatment of an ultrasonic probe of 15 minutes‘
sonication time and 20% amplitude. While the highest recovery of TPC of
245.780±9.858 mg GAE/g hull powder was obtained using ethanol 0.20% HCl acidified
aqueous solvent by pretreating the mangosteen sample with ultrasonic probe conditions
of 25 minutes sonication time and 80% amplitude.
In conclusion, methanol has been found to be the best extraction solvent in giving the
highest TPC yield. The optimum extraction conditions of 2 hours extraction time, 0.05
solid to solvent ratio and 69.77% methanol concentration obtained from RSM gave the
maximum TPC yield 140.66 mg GAE/g hull powder with an improvement of 85.41%
compared to extraction with 20 hours extraction time, 0.20 solid to solvent ratio and
pure methanol solvent. The extraction solvent acidified with 20 ml of Mexican lime
juice gave higher TMA yield compared to HCl acidified‘s. The ultrasonication of 15
minutes and 20% amplitude gave the highest TMA recovery of 2.917±0.039 mg cy-3-
glu/g hull powder using acidified methanol solvent, while 25 minutes and 80%
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amplitude gave the highest TPC of 245.780±9.858 mg GAE/g hull powder using
acidified ethanol solvent. Overall, the extraction of TPC and TMA yields from
mangosteen hull have been optimized by reducing the extraction time, solid to solvent
ratio using acidified extraction solvent with ultrasonic wave treatment.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
PENGEKSTRAKAN KANDUNGAN JUMLAH FENOL DAN JUMLAH
MONOMERIK ANTOSIANIN DARIPADA KULIT MANGGIS (GARCINIA
MANGOSTANA L.)
Oleh
CHEOK CHOON YOONG
Julai 2012
Pengerusi : Chin Nyuk Ling, PhD
Fakulti : Kejuruteraan
Manggis ialah buah-buahan tropika tempatan yang popular kerana isinya adalah lazat
dan mempunyai nilai zat dan mineral yang tinggi. Walau bagaimanapun, manggis kulit
dibuangkan sebagai bahan buangan oleh orang-orang tempatan kerana rasa kepahitan
yang tidak menyenangkan, walaupun ia terkandung jumlah fenolik dan antosianin yang
mempunyai banyak sifat farmaseutikal yang penting. Disebabkan hubungan antara
kandungan jumlah fenol (TPC) dan antosianin (TMA) dengan kapasiti antioksidan yang
ketara, satu siri eksperimen telah dijalankan untuk mempertingkatkan hasil ekstrak
manggis mentah yang dinilai dari segi kandungan jumlah fenol (TPC) dan antosianin
(TMA). Kajian ini melibatkan eksperimen dari asas ke pengoptimuman, berdasarkan
hipotesis bahawa metanol, pelarut berasid, dan rawatan gelombang ultrasonik boleh
memperolehi hasil pengekstrakan yang lebih tinggi. Keputusan dinyatakan dalam mg
asid gallic/g serbuk kulit untuk TPC, manakala bagi TMA, dinyatakan dalam mg cy-3-
glu/g serbuk kulit.
Kesan pelarut, i.e. etil asetat, isopropanol, aseton, etanol, metanol dan air suling dikaji
terhadap pengekstrakan jumlah kandungan fenol (TPC) daripada kulit manggis. Metanol
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telah didapati sebagai pelarut yang paling berkesan dalam memberi pulangan tertinggi (p
< 0.05) TPC, berbanding dengan etanol, aseton, isopropanol, etil asetat dan air suling.
Persamaan regresi dengan pekali penentuan yang tinggi (R2 0.990) telah diperolehi
secara langsung daripada serapan UV-Vis spektrofotometer untuk ekstrak aseton, etanol,
dan metanol dengan kaedah Folin-Ciocalteu (FC), menunjukkan persamaan regresi yang
diperolehi ini boleh digunakan untuk menentukan TPC tanpa menggunakan kaedah FC.
Keadaan optimum iaitu masa pengekstrakan, nisbah pepejal kepada pelarut, dan
kepekatan metanol untuk pengekstrakan jumlah fenol daripada serbuk kulit manggis
telah dikaji dengan menggunakan kaedah gerak balas permukaan (RSM). Data
eksperimen yang diperolehi diisikan ke dalam model polinomial tertib kedua dengan R2
0.897. Parameter pengekstrakan optimum yang memberi hasil TPC yang maksimum
iaitu 140.66 mg GAE/g serbuk kulit adalah dari keadaan pengekstrakan 2 jam, 0.05
nisbah pepejal kepada pelarut dan kepekatan metanol pada 69.77%. Analisis
menggunakan rangkaian neural tiruan (ANN) meramalkan data menunjukkan nilai R2
yang lebih tinggi iaitu 0.945 dan nilai purata sisihan mutlak 4.01% berbanding 5.37%
bagi RSM. Ini mencadangkan bahawa ANN adalah teknik pemodelan yang lebih baik
berbanding dengan RSM bagi data tak linear dalam ramalan hasil TPC yang
diekstrakkan daripada serbuk kulit manggis.
Pemulihan antosianin daripada kulit manggis telah dikaji menggunakan pelarut akueus
berasid dengan jus limau nipis (C. aurantifolia Swingle) berbanding dengan pelarut
tradisi iaitu metanol berasid HCl. Dengan tambahan 0.20 ml jus limau nipis/ml
pengekstrakan mengunakan pelarut metanol akueus memberikan maksimum jumlah
monomerik antosianin (TMA) sebanyak 4.742±0.590 mg cy-3-glu/g serbuk kulit
berbanding dengan 2.950±0.265 mg cy-3-glu/g serbuk kulit apabila menggunakan
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pelarut pengekstrakan yang berasid dengan 0.20% HCl. Peningkatan yang ketara (p <
0.05) antosianin sebanyak 60.75% mencadangkan bahawa jus limau nipis semulajadi ini
adalah ejen asid yang baik dan boleh menggantikan HCl dalam mengasidkan pelarut.
Serbuk kulit manggis dengan saiz partikel antara 250 m hingga 500 m juga didapati
memberi pemulihan antosianin tertinggi (p < 0.05).
Dua jenis pelarut pengekstrakan, iaitu, pelarut metanol dan etanol akueus berasid dengan
0.20% HCl telah digunakan untuk mengkaji kesan masa ultrasonik dan amplitud
terhadap TMA dan TPC daripada kulit manggis. Pulangan TMA tertinggi sejumlah
2.917±0.039 mg cy-3-glu/g serbuk kulit telah dicapai dengan menggunakan pelarut
metanol akueus berasid dengan 0.20% HCl dengan ultrasonik prapengolahan selama 15
minit dan 20% amplitud manakala pulangan TPC tertinggi sebanyak 245.780±9.858 mg
GAE/g serbuk kulit telah diperolehi menggunakan pelarut etanol akueus berasid dengan
0.20% HCl selama 25 minit dan 80% amplitud.
Kesimpulannya, metanol telah didapati sebagai pelarut pengekstrakan yang terbaik
dalam memberikan hasil TPC tertinggi. Keadaan pengekstrakan optimum 2 jam masa
pengekstrakan, 0.05 nisbah pepejal kepada pelarut dan 69.77% kepekatan metanol
diperolehi dari RSM memberi hasil TPC maksimum 140,66 mg GAE/g serbuk kulit
dengan peningkatan sebanyak 85.41% berbanding pengekstrakan dengan 20 jam masa
pengekstrakan, 0.20 nisbah pepejal kepada pelarut dan metanol pelarut tulen. Pelarut
pengekstrakan berasid dengan 20 ml jus limau nipis memberi hasil TMA yang lebih
tinggi berbanding dengan HCl berasid. Ultrasonikasi 15 minit dan 20% amplitud
memberi pulangan TMA tertinggi 2.917±0.039 mg cy-3-glu/g serbuk kulit apabila
menggunakan pelarut metanol berasid, manakala 25 minit dan 80% amplitud memberi
pulangan TPC tertinggi 245.780±9.858 mg GAE/g serbuk kulit apabila menggunakan
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pelarut etanol berasid. Secara keseluruhan, pengekstrakan hasil TPC dan TMA dari
manggis hull telah dioptimumkan dengan mengurangkan masa pengekstrakan, nisbah
pepejal kepada pelarut menggunakan pelarut pengekstrakan berasid dengan rawatan
gelombang ultrasonik.
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ACKNOWLEDGEMENTS
I am grateful to my supervisor Associate Prof. Ir. Dr. Chin Nyuk Ling, for her patience
in guiding me to organize my thoughts and works concisely in this research. Her open-
minded and meticulous guidance made this research progress smoothly, and be finally
completed.
I am also deeply thankful to my co-supervisor Prof. Dr. Law Chung Lim, at University
of Nottingham Malaysia Campus, for providing the laboratory facilties for this entire
research. I take this opportunity to thank Associate Prof. Dr. Yus Aniza and Dr. Rosnita,
also members of my supervisory committee, who spared valuable time in sharing views
and offering suggestions for this research.
I am greatly indebted to En. Raman bin Morat, the laboratory technician, for his
assistance in providing all the well maintained equipment and apparatus needed for this
research. Finally, I wish to express my gratitute to UPM for granting Graduate Reseach
Fellowship for my entire PhD program.
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I certify that an Examination Committee has met on 19 July 2012 to conduct the final
examination of Cheok Choon Yoong her thesis entitled "Extraction of Total Phenolic
Content and Total Monomeric Anthocyanin from Mangosteen (Garcinia Mangostana L.)
Hull" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and
Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee
recommends that the student be awarded the Doctor of Philosophy.
Members of the Examination Committee were as follows:
Siti Mazlina Mustapa Kamal, PhD
Associate Professor Faculty of Engineering
Universiti Putra Malaysia
(Chairman)
Norhafizah Abdullah, PhD
Associate Professor Faculty of Engineering
Universiti Putra Malaysia
(Internal Examiner)
Russly Abdul Rahman, PhD
Professor
Institut Penyelidikan Produk Halal
Universiti Putra Malaysia
(Internal Examiner)
Muthupandian Ashokkumar, PhD
Associate Professor
School of Chemistry
University of Melbourne
Australia
(External Examiner)
_____________________________
SEOW HENG FONG, PhD Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted
as fulfilment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Ir. Chin Nyuk Ling, PhD
Associate Professor
Food and Process Engineering
Faculty of Engineering
Universiti Putra Malaysia
(Chairman)
Yus Aniza Yusof, PhD
Lecturer
Food and Process Engineering
Faculty of Engineering
Universiti Putra Malaysia
(Member)
Rosnita A Talib, PhD
Lecturer
Food and Process Engineering
Faculty of Engineering
Universiti Putra Malaysia
(Member)
Law Chung Lim, PhD
Professor
Department of Chemical Engineering
Faculty of Engineering
University of Nottingham Malaysia Campus
(Member)
_______________________
BUJANG KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 13 December 2012
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DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and
citations which have been duly acknowledged. I also declare that it has not been
previously or concurrently submitted for any other degree at UPM or other institutions.
_______________________
CHEOK CHOON YOONG
Date: 19 July 2012
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TABLE OF CONTENT
Page
ABSTRACT ii
ABSTRAK vi
ACKNOWLEDGEMENTS x
APPROVAL xi
DECLARATION xiii
LIST OF TABLES xviii
LIST OF FIGURES xx
LIST OF ABBREVIATIONS xxii
CHAPTER
1 INTRODUCTION 1
1.1 Research Background 1
1.2 Problem Statement 2
1.3 Research Hyphothesis 4
1.4 Objectives 5
1.5 Thesis Layout 6
2 LITERATURE REVIEW 7
2.1 Introduction 7
2.2 Background Review of Mangosteen 8
2.2.1 Nutritional Values, Pharmaceutical Properties, and
Other Usages of Mangosteen (Garcinia mangostana L.) 11
2.3 Phenolic Compounds of Plant Materials 14
2.4 Phenolic Compounds Isolated and Identified from
Mangosteen 15
2.5 Methods of Phenolic Compounds Quantification 18
2.5.1 Comparison Studies between Spectrophotometric
and Chromatographic Techniques 20
2.5.2 Folin‒Ciocalteu (FC) Method 21
2.5.3 pH Differential Method 24
2.5.4 Relationship between TPC or Anthocyanins with
Antioxidant Properties 27
2.6 Extraction of Phenolic Compounds 28
2.6.1 Extraction Techniques of Phenolic Compounds from
Plant Materials 28
2.6.2 Maceration Solvent Extraction 30
2.6.3 Extraction Solvent Used in Extracting Phenolic
Compounds from Mangosteen 36
2.6.4 Extraction of Anthocyanins from Plant Materials 39
2.6.5 Citrus Juice as an Acidic Agent in Extraction of
Bioactive Compounds from Plant Materials 39
2.7 Effects of Solvent on Bioactive Compounds Yield 42
2.7.1 Influence of Solvent Type or Solvent Concentration
on TPC 42
2.7.2 Influence of Solvent on Anthocyanins Yield 44
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2.8 Ultrasound-Assisted Extraction (UAE) 46
2.8.1 Introduction 46
2.8.2 Propagation of Cavitation Bubble under Ultrasound
Power 46
2.8.3 Applications of Ultrasonic in Bioactive Compound
Extraction 48
2.8.4 Applications in Other Food Researches 49
2.8.5 Performance of Ultrasonic-Assisted Extraction 51
2.9 Optimization Techniques and Tools in Food Researches 56
2.9.1 Response Surface Methodology (RSM) 56
2.9.2 Artificial Neural Networks (ANN) 61
2.9.3 Comparison Studies between RSM and ANN 62
2.10 Summary 63
3 MATERIALS AND METHODS 64
3.1 Introduction 64
3.2 Extraction of Total Phenolics from Mangosteen Hull
Powder Using Different Types of Solvents 66
3.2.1 Experimental Solvents and Chemicals 66
3.2.2 Collection and Preparation of Dried Mangosteen
Hull 66
3.2.3 Preparation of Mangosteen Hull Powder 67
3.2.4 Mangosteen Crude Extract Preparation 68
3.2.5 Determination of Total Phenolic Content (TPC)
Using Folin‒Ciocalteu (FC) Method 69
3.2.6 Establishing a Relationship of Total Phenolics and
Direct UV‒Vis Spectrophotometer Absorbance of
Crude Mangosteen Extract 75
3.3 Optimization of Total Phenolic Content (TPC) Extracted
from Mangosteen Hull Powder Using Response Surface
Methodology (RSM) versus Artificial Neural Networks
(ANN) 76
3.3.1 Mangosteen Hull Powder and Crude Extract
Preparation 76
3.3.2 TPC Determination as Variable Response 77
3.3.3 Screening Study and Design of Experiments for
RSM Modelling 78
3.3.4 Artificial Neural Networks (ANN) Modelling 83
3.4 Effects of Mexican Lime Juice Acidified Methanol
Aqueous Extraction Solvent on Anthocyanins Recovery
from Mangosteen Hull Powder 83
3.4.1 Mangosteen Hull Powder Preparation 83
3.4.2 Preparation of Mexican Lime Juice and HCl
Acidified Methanol Aqueous Extraction Solvents 83
3.4.3 Determination of Organic Acids in Mexican Lime
Juice 85
3.4.4 Extraction of Anthocyanins 86
3.4.5 Total Monomeric Anthocyanin (TMA)
Determination 86
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3.5 Influence of Different Mangosteen Hull Powder Particle
Sizes on TMA Yield 87
3.6 Optimization of Ultrasound-Assisted Extraction of Total
Monomeric Anthocyanin (TMA) and Total Phenolic
Content (TPC) from Mangosteen Hull using Full Factorial
Design 88
3.6.1 Mangosteen Hull Preparation 88
3.6.2 Full Factorial Design for Solvent Extraction
Coupled With Ultrasonic Treatment 89
3.6.3 Total Monomeric Anthocyanin (TMA) and Total
Phenolic Content (TPC) Determination 91
3.6.4 Comparison between Ultrasonic Probe and Bath
Pretreated Mangosteen Sample on TMA and TPC
Yields 92
3.7 Statistical Analysis 93
4 RESULTS AND DISCUSSION 95
4.1 Introduction 95
4.2 Extraction of Total Phenolic Content (TPC) From
Mangosteen hull 96
4.2.1 Effect of Solvents on TPC 96
4.2.2 Relationship between Direct UV‒Vis
Spectrophotometer Absorbance and FC Methods
For TPC 99
4.3 Optimization of Total Phenolic Content (TPC) Extracted
from Mangosteen Hull using Response Surface
Methodology (RSM) Versus Artificial Neural Networks
(ANN) 103
4.3.1 Experimental Ranges from Screening Study 103
4.3.2 Modelling and Optimization using RSM 105
4.3.3 ANN Prediction on TPC Yield 110
4.4 Anthocyanins Recovery from Mangosteen Hull using
Mexican Lime Juice Acidified Aqueous Solvent Extraction 112
4.4.1 Effect of Mexican Lime Juice on Total Monomeric
Anthocyanin (TMA) Yield Compared to HCl
Acidified Methanol Aqueous Extraction Solvents 112
4.4.2 Effect of Extraction Solvent pH on TMA Yield 117
4.5 Effect of Different Particle Sizes on Total Monomeric
Anthocyanin (TMA) Yield 119
4.6 Optimization of Ultrasound-Assisted Extraction of Total
Monomeric Anthocyanin (TMA) and Total Phenolic
Content (TPC) from Mangosteen Hull using Full Factorial
Design 121
4.6.1 Effect of Ultrasonic Sonication Time and Amplitude
on TMA Yield 121
4.6.2 Effect of Ultrasonic Sonication Time and Amplitude
on TPC Yield 124
4.6.3 Comparison of Ultrasonic Probe and Bath with
Conventional Magnetic Stirring Extraction of TMA
and TPC from Mangosteen Hull Powder 127
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4.7 Engineering Aspect in Productivity of Extracting TPC and
TMA from Mangosteen Hull 132
5 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
FOR FUTURE RESEARCH 137
REFERENCES 139
APPENDICES 172
BIODATA OF STUDENT 195
LIST OF PUBLICATIONS 196
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