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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