Waxes, policosanols and aldehydes in sugarcane (Saccharum officinarum L.) and okinawan brown sugar (kokuto)

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WAXES, POLICOSANOLS AND ALDEHYDES IN SUGARCANE (Saccharum officinarum L.) AND OKINAWAN BROWN SUGAR

(KOKUTO)

YONATHAN ASIKIN

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY BOGOR

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DECLARATION OF ORIGINALITY

I hereby declare that this thesis is my own work and to the best of my knowledge it contains no material previously published or written by another person, nor materials which to a substantial extent has been accepted for the award of any degree or diploma at any university, except where due acknowledgement has been made.

Bogor, August 2008

Yonathan Asikin

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ABSTRACT

YONATHAN ASIKIN. Waxes, Policosanols and Aldehydes in Sugarcane

(Saccharum officinarum L.) and Okinawan Brown Sugar (Kokuto). Under

direction of RIZAL SYARIEF and HANIFAH NURYANI LIOE.

Waxes, long chain alcohols and aldehydes were found in several variants of sugarcane (Saccharum officinarum L.) and Kokuto, a non-centrifuged Okinawan cane brown sugar. Long chain alcohols, policosanols, have been reported to have beneficial effect to human health. The composition of wax in sugarcane was analyzed using HPLC with an evaporative light scattering detector. Sugarcane wax composed of 55–60% aldehydes, sterol esters and wax esters, 32–40% alcohols, and small amounts of triacylglycerols, acids and sterols. Extraction of policosanols performed effectively with hexane and methanol (20:1 v/v), while that of long chain aldehydes was with chloroform and methanol (2:1 v/v). Their composition was determined using GC with a flame ionization detector, whereas their compounds were identified using GC-MS. Sugarcane rinds contained up to 500 mg policosanols and 600 mg aldehydes per 100 g sample of Ni 22 cultivar. The content of policosanol and long chain aldehyde in Kokuto was influenced by its production systems. Compositional analysis of the end product confirmed the presence of policosanols and aldehydes up to 85 mg and 8 mg respectively per 100 g sample of Kokuto A, a product of brown sugar manufacture with open pan heating system. Octacosanol and octacosanal were found to be the major wax components in both sugarcane and Kokuto samples. This study revealed significant difference in content and composition of waxes, policosanols and long chain aldehydes between sugarcane parts, cultivars and harvesting times; also between Kokuto types and production methods.

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RINGKASAN

YONATHAN ASIKIN. Wax, Polikosanol dan Aldehida pada Tebu (Saccharum

officinarum L.) dan Gula Coklat Okinawa (Kokuto). Dibimbing oleh RIZAL

SYARIEF dan HANIFAH NURYANI LIOE.

Wax tebu (Saccharum officinarum L.) telah menjadi perhatian bagi banyak orang, karena aplikasinya pada berbagai bidang industri dan sifat fungsional dari salah satu senyawa yang terkandung di dalamnya, yaitu alkohol rantai panjang. Senyawa alkohol rantai panjang (C20–C30) atau polikosanol telah banyak diteliti dan dilaporkan terkait dengan dampak positifnya bagi kesehatan manusia seperti menurunkan agregasi platelet, menurunkan kadar LDL dalam darah, dan menghambat sintesis kolesterol. Dalam penelitian ini kandungan dan komposisi kimiawi wax, polikosanol dan aldehida rantai panjang diteliti dari beberapa varietas tebu dan Kokuto, gula coklat non-sentrifugasi dari Okinawa, Jepang.

Tujuan penelitian ini adalah untuk mengetahui komposisi wax, polikosanol dan aldehida rantai panjang dengan analisis TLC, HPLC-ELSD, FID dan GC-MS. Secara khusus, analisis ditujukan untuk mengetahui pengaruh metode dan waktu ekstraksi; pengaruh bagian-bagian batang tebu, varietas dan umur panen tebu; serta pengaruh cara produksi dan jenis Kokuto terhadap kandungan wax, polikosanol dan aldehida rantai panjang pada tebu dan Kokuto.

Sampel yang digunakan dalam penelitian ini adalah bagian-bagian batang tebu varietas Ni 15, bagian kulit dari tujuh varietas tebu (Ni 13, Ni 17, Ni 22, NiF 8, NCo 310, F 161 dan F 177), serta tujuh jenis Kokuto (tipe A–G). Komposisi wax tebu dianalisis secara kualitatif dengan teknik TLC dan dikuantifikasi dengan HPLC yang dilengkapi detektor evaporative light scattering. Kandungan dan komposisi senyawa polikosanol dan aldehida rantai panjang dianalisis oleh GC dengan detektor flame ionization, sedangkan struktur senyawanya diidentifikasi oleh GC-MS. Pengaruh setiap percobaan dianalisis secara statistik dengan rancangan acak lengkap satu faktor.

Studi ini memberikan informasi mengenai senyawa fungsional polikosanol yang terkandung pada tebu dan gula coklat. Polikosanol dapat diekstrak secara efektif dengan pelarut heksana dan metanol (20:1 v/v), sedangkan aldehida rantai panjang dengan pelarut kloroform dan metanol (2:1 v/v). Identifikasi senyawa polikosanol dengan GC-MS terlihat pada pola fragmen massa trimetilsilil-eter dari ion target, sedangkan senyawa aldehida rantai panjang teridentifikasi dari pecahan fragmen spesifik dari senyawa aldehida. Oktakosanol dan oktakosanal merupakan komponen penyusun utama dari wax tebu dan Kokuto.

Studi ini mengungkapkan bahwa bagian-bagian tebu, varietas dan umur panen tebu berpengaruh nyata terhadap komposisi dan kandungan wax, polikosanol dan aldehida rantai panjang pada tebu. Komposisi wax tebu yang dianalisis dengan HPLC-ELSD adalah 55–60% campuran senyawa aldehida, sterol ester dan wax ester, 32–40% alkohol, dan sejumlah kecil triasilgliserol, asam dan sterol.

Senyawa polikosanol dan aldehida rantai panjang pada bagian kulit tebu yang dipisahkan dengan Cane Separation System ditemukan lebih banyak dibandingkan dengan bagian terkupasnya, yakni dengan 80 mg per 100 g sampel

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tebu varietas Ni 15, sedangkan bagian terkupasnya tersebut hanya mengandung sekitar 1 mg polikosanol dan aldehida per 100 g sampel. Seluruh bagian batang tebu diketahui mengandung 35 mg polikosanol dan 24 mg aldehida per 100 g sampel.

Pengupasan kulit tebu secara manual membuat wax epidermis masih melekat pada sampel, sehingga total polikosanol dan aldehida yang terkandung di dalamnya mencapai 500 mg dan 600 mg per 100 g sampel tebu varietas Ni 22. Total kandungan wax pada kulit tebu tersebut dipengaruhi oleh jenis varietas tebu, kondisi pertumbuhan dan umur tanaman tersebut. Kandungan senyawa aldehida pada kulit tebu meningkat lebih besar dibandingkan dengan senyawa polikosanol sejalan dengan meningkatnya umur tanaman terebut.

Pada gula coklat Kokuto, kandungan polikosanol dan aldehida rantai panjang dipengaruhi oleh sistem produksinya. Kokuto A yang diproduksi dengan sistem pemanasan wadah terbuka didapati mengandung polikosanol dan aldehida paling tinggi dengan 85 mg dan 8 mg per 100 g sampel. Sampel Kokuto tipe lainnya diproduksi dengan teknologi pemanasan vakum yang mensyaratkan proses filtrasi yang mengakibatkan pemisahan sejumlah komponen wax dan molases dari sirup gula dan produk akhirnya, sehingga kandungan polikosanol dan aldehida lebih sedikit dibandingkan Kokuto A. Dengan ini didapati bahwa cara produksi dan jenis Kokuto berpengaruh nyata terhadap komposisi dan kandungan polikosanol dan aldehida rantai panjang pada Kokuto.

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© Copyright of this thesis belongs to Bogor Agricultural University (Institut Pertanian Bogor), Indonesia and University of the Ryukyus, Japan, 2008.

1. Due acknowledgement must always be made of the use of any material contained in, or derived from, this thesis.

a. Any person, may however, use content of the thesis for the purposes of education, private study, research, scientific report or review.

b. The use of any material contained in, or derived from, this thesis must not make negative effect for the University.

2. The written permission from the University is required before any part of the thesis may be copied for any other purpose.

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WAXES, POLICOSANOLS AND ALDEHYDES IN SUGARCANE (Saccharum officinarum L.) AND OKINAWAN BROWN SUGAR

(KOKUTO)

YONATHAN ASIKIN

Thesis

as partial fulfillment of the requirements for the degree of Master of Science

in the Department of Food Science and Technology

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY BOGOR

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Title of Thesis : Waxes, Policosanols and Aldehydes in Sugarcane (Saccharum officinarum L.) and Okinawan Brown Sugar (Kokuto)

Name : Yonathan Asikin Student ID : F251050111

Approved by Advisory Committee

Prof. Dr. Ir. Rizal Syarief, DESS

Chair Member

Dr. Ir. Hanifah Nuryani Lioe, M.Si.

Acknowledged by

Head of Food Science Dean of Graduate School Study Program

Dr. Ir. Ratih Dewanti, M.Sc. Prof. Dr. Ir. Khairil Anwar Notodiputro, M.S.

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PREFACE

I would like to thank God, Lord Jesus Christ, for his mercy and spirit throughout my study and thesis work in Graduate School of Bogor Agricultural University, Indonesia. He always helps and protects me. I can do everything through Christ who strengthens me.

This thesis owes its existence to the help, support and inspiration of many people. Firstly, I would like to express my sincere appreciation to Prof. Dr. Rizal Syarief as Chair of Advisory Committee for his support and encouragement during my study in Bogor Agricultural University. I am very grateful to Dr. Hanifah Nuryani Lioe as Member of Advisory Committee for her advice and supervision during the thesis work. I am also indebted to Dr. Feri Kusnandar as Non-committee Examiner for his constructive comments on this thesis.

I would like to thank University of the Ryukyus, Japan for the Short Term Regular Program (STRP) and for giving me the opportunity to conduct my research in Laboratory of Food Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus; and the Japan Student Service Organization (JASSO) for the scholarship.

I would like to express my sincere appreciation to my supervisor during STRP period, Prof. Dr. Koji Wada, Head of Laboratory of Food Chemistry, Faculty of Agriculture, University of the Ryukyus for his advice and support throughout the research. I gratefully thank Dr. Kensaku Takara for valuable discussions during my research work. I also would like thank Okinawa Prefectural Agricultural Research Center, Japan for providing sugarcane and Kokuto samples.

I wish to thank all lecturers and colleagues, especially IPN 2005, at the Food Science Study Program of Graduate School of Bogor Agricultural University. It has been a pleasure to work with you.

Last but not least, many thanks to my parents, brother and sisters for their true and endless love, for never-failing patience and encouragement.

Bogor, August 2008

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WAXES, POLICOSANOLS AND ALDEHYDES IN SUGARCANE (Saccharum officinarum L.) AND OKINAWAN BROWN SUGAR

(KOKUTO)

YONATHAN ASIKIN

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY BOGOR

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DECLARATION OF ORIGINALITY

Bogor, August 2008

Yonathan Asikin

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ABSTRACT

YONATHAN ASIKIN. Waxes, Policosanols and Aldehydes in Sugarcane

(Saccharum officinarum L.) and Okinawan Brown Sugar (Kokuto). Under

direction of RIZAL SYARIEF and HANIFAH NURYANI LIOE.

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RINGKASAN

YONATHAN ASIKIN. Wax, Polikosanol dan Aldehida pada Tebu (Saccharum

officinarum L.) dan Gula Coklat Okinawa (Kokuto). Dibimbing oleh RIZAL

SYARIEF dan HANIFAH NURYANI LIOE.

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© Copyright of this thesis belongs to Bogor Agricultural University (Institut Pertanian Bogor), Indonesia and University of the Ryukyus, Japan, 2008.

1. Due acknowledgement must always be made of the use of any material contained in, or derived from, this thesis.

a. Any person, may however, use content of the thesis for the purposes of education, private study, research, scientific report or review.

b. The use of any material contained in, or derived from, this thesis must not make negative effect for the University.

2. The written permission from the University is required before any part of the thesis may be copied for any other purpose.

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WAXES, POLICOSANOLS AND ALDEHYDES IN SUGARCANE (Saccharum officinarum L.) AND OKINAWAN BROWN SUGAR

(KOKUTO)

YONATHAN ASIKIN

Thesis

as partial fulfillment of the requirements for the degree of Master of Science

in the Department of Food Science and Technology

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY BOGOR

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Title of Thesis : Waxes, Policosanols and Aldehydes in Sugarcane (Saccharum officinarum L.) and Okinawan Brown Sugar (Kokuto)

Name : Yonathan Asikin Student ID : F251050111

Approved by Advisory Committee

Prof. Dr. Ir. Rizal Syarief, DESS

Chair Member

Dr. Ir. Hanifah Nuryani Lioe, M.Si.

Acknowledged by

Head of Food Science Dean of Graduate School Study Program

Dr. Ir. Ratih Dewanti, M.Sc. Prof. Dr. Ir. Khairil Anwar Notodiputro, M.S.

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PREFACE

I wish to thank all lecturers and colleagues, especially IPN 2005, at the Food Science Study Program of Graduate School of Bogor Agricultural University. It has been a pleasure to work with you.

Last but not least, many thanks to my parents, brother and sisters for their true and endless love, for never-failing patience and encouragement.

Bogor, August 2008

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CURRICULUM VITAE

Yonathan Asikin was born in Karawang, West Java Province, Republic of Indonesia, on November 24, 1981. After graduating from Karawang State Senior High School (SMUN 1 Karawang) in 1999, he entered Bogor Agricultural University, Indonesia, from 1999 to 2003 where he obtained a Bachelor (Sarjana) of Agricultural Technology degree in Food Technology in 2003.

He entered Graduate School of Bogor Agricultural University, majoring food science (Master Program), in 2005. During school he attended Short Term Regular Program (STRP) in University of the Ryukyus, Japan, from April 2007 to March 2008 as research student. He conducted his thesis research in Laboratory of Food Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus.

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Page

LIST OF TABLES ... xi LIST OF FIGURES ... xii LIST OF APPENDIX ... xiv LIST OF ABBREVIATIONS ... xv INTRODUCTION... 1 LITERATURE REVIEW ... 4 Sugarcane (Saccharum officinarum L.) ... 4 Brown Sugar ... 7 Plant Wax ... 8 Wax Compositional Analysis... 10 Policosanol in Human Health ... 14 MATERIALS AND METHODS ... 17 Materials... 17 Methods ... 19 Wax compositional analysis by TLC and HPLC-ELSD ... 21 Policosanol and long chain aldehyde analysis by GC-FID ... 24 Policosanol and long chain aldehyde identification by GC-MS ... 27 Statistical analysis ... 28 RESULTS AND DISCUSSION ... 30 Sugarcane Wax Composition ... 30 GC Chromatogram and Mass Spectrum of Policosanols and Long Chain Aldehydes... 38 Policosanols and Long Chain Aldehydes in Sugarcane Rind ... 43 Policosanols and Long Chain Aldehydes in Kokuto ... 50 CONCLUSION ... 57 REFERENCES ... 59 APPENDIX ... 65

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LIST OF TABLES

Page

1 Scientific classification of sugarcane ... 4 2 Sugarcane producer countries in 2005 ... 5 3 Mixture composition of aliphatic alcohols (policosanols) ... 12 4 Content and composition of policosanols in some materials ... 13 5 Wax compositions of rind part of Okinawan sugarcane cultivars, obtained by

HPLC-ELSD ... 34 6 Mass fragmentation pattern of policosanols and long chain aldehydes ... 42 7 Policosanol and long chain aldehyde contents of rind part of Okinawan

sugarcane cultivars ... 45 8 Policosanol and long chain aldehyde contents of Kokuto (A–G) and brown

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xii

LIST OF FIGURES

Page

1 Kokuto, Okinawan brown sugar ... 8

2 Rind (a) and (b) pith parts of sugarcane separated by CSS ... 17 3 Cane Separation System (CSS) ... 18 4 Experimental designs of sugarcane wax, policosanol and long chain aldehyde

analysis ... 19 5 Production lines of Kokuto ... 20

6 Experimental designs of policosanol and long chain aldehyde analysis in

Kokuto ... 21

7 Thin layer chromatography of waxes of several sugarcane cultivars ... 30 8 HPLC chromatogram of sugarcane rind of Ni 15 cultivar ... 32 9 Standard curve of triacylglycerol ... 32 10 HLPC chromatograms of wax composition standards ... 33 11 Policosanol and long chain aldehyde contents in sugarcane Ni 15 cultivar,

obtained by GC-FID... 36 12 Typical gas chromatogram of (a) policosanol and long chain aldehyde

standards, (b) Kokuto A, (c) sugarcane rind of Ni 15 cultivar ... 39

13 Standard curve of octacosanol ... 40 14 Mass spectrum of trimethylsilyl ether of C28 of standard ... 41 15 Mass spectrum of trimethylsilyl ether of C28 of Kokuto ... 41

16 Mass spectrum of C28 aldehyde of sugarcane rind of standard ... 42 17 Mass spectrum of C28 aldehyde of sugarcane rind of Ni 15 cultivar ... 42 18 Influence ofsoxhlettimes on policosanol and long chain aldehyde extraction

of sugarcane rind of Ni 15 cultivar ... 43 19 Composition of (a) policosanols and (b) long chain aldehydes in sugarcane

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xiii 20 Comparison of (a) policosanol and (b) long chain aldehydes contents in 9 and

12 months peeled sugarcane rind samples ... 47 21 Product derivates from sugarcane ... 49 22 Influence of methods and solvent types on policosanol and long chain

aldehyde extraction of Kokuto A ... 51

23 Influence ofsoxhlettimes on policosanol and long chain aldehyde extraction of Kokuto A ... 52

24 Composition of (a) policosanols and (b) long chain aldehydes in Kokuto A . 53

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xiv

LIST OF APPENDIX

Page

1 Mass spectrum of trimethylsilyl derivates of policosanols ... 66 2 Mass spectrum of long chain aldehydes ... 69 3 Statistical analysis of total policosanol content in sugarcane rind, obtained by

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LIST OF ABBREVIATIONS

Abbreviation Term

CSS Cane Separation System

ELSD Evaporative Light Scattering Detector

HPLC High Performance Liquid Chromatography

GC Gas Chromatography

GC-FID Gas Chromatography Flame Ionization Detector

GC-MS Gas Chromatography Mass Spectra

LDL Low Density Lipoprotein

MSTFA N-Methyl-N-(trimethylsilyl) trifluoroacetamide MTBE Methyl tert-butyl ether

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I. INTRODUCTION

Sugarcane (Saccharum officinarum L.) wax has been a matter of interest, due to its industry application and functionality of one of its compound, long chain alcohols. The surfaces of plants, including sugarcane, are coated with several layers of lipophilic material, the outermost being the epicuticular wax. It serves many purposes, for example to limit the diffusion of water and solutes, while permitting a controlled release of volatiles that may deter pests or attract pollinating insects. It also provides protection from diseases and insects, and helps the plants resist drought.

Long chain alcohols, which is well-known as policosanols, is a group of long chain (C20–C30) aliphatic primary alcohols which is of a great interest due to their health beneficial effect for human health, such as reducing platelet aggregation, reducing low-density lipoprotein levels in blood, inhibiting cholesterol synthesis, and ergogenic properties (Castano et al. 2003; Singh et al. 2006; Taylor et al. 2003). Long chain aldehydes as well as alcohols are one of main component of natural wax extracted from plant (Adhikari et al. 2006). Straight chain aldehyde also was known as one of lipid biomarker in leaves and roots of plant (Jansen et al. 2006).

Recently, research in policosanol analysis with several kinds of materials and techniques have been well reported (Adhikari et al. 2006; Wang et al. 2007; Wu et al. 2007). Sugarcane and its wax have been reported contain a number of policosanols used as major source in commercial product of policosanol (Irmak et al. 2006; Morrison et al. 2006; Nuissier et al. 2002). However, only a few information of long chain aldehyde analysis are published, especially for

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sugarcane and its products.

Kokuto, a unique brown cane sugar, has been traditionally produced in Okinawa, Japan from sugarcane by non-centrifugal method, without molasses removing process. This product has been reported to contain some antioxidants and phenolic compounds (Takara et al. 2002, 2003). It is then expected that Kokuto as one of cane food product contain much wax components, including policosanol which has beneficial health effect as described above.

Indonesian sugar industry, back to the seventeenth century, was known as one of the oldest and biggest sugar industry in the world. It reached its zenith in the early-thirties when 179 factories produced nearly 3 million MT of sugar annually. Following several up and down conditions in many periods of times, yet, since 1967, Indonesia has reverted to a net sugar importer position and since the mid-eighties imports have continued to rise. The present average cane yields are thus about 7.5% (Hadisaputro et al. 2008). Thus this study would explored information of functional compounds potentially contained in sugarcane and brown sugar. Sugarcane might be potent sources for high value added products of sugarcane derivates, such as cane wax and policosanol.

The main purpose of this study is to determine wax, policosanol and aldehyde compositions in sugarcane and Kokuto, Okinawan brown sugar, with TLC, HPLC-ELSD, GC-FID and GC-MS. Some specific aims were applied in this study, i.e.:

a. to determine the effect of extraction methods and times on the policosanol and long chain aldehyde contents of sugarcane rinds and Kokuto,

b. to determine the effect of sugarcane cultivars on wax, policosanol and long chain aldhyde contents and compositions of the sugarcane rinds,

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c. to determine policosanol and long chain aldehyde contents in different parts of sugarcane,

d. to determine the effect of sugarcane harvesting time on the policosanol and long chain aldehyde contents of the sugarcane rinds,

e. to determine the effect of Kokuto types on the policosanol and long chain aldehyde contents of the Kokuto, and

f. to determine the effect of Kokuto production types on the policosanol and long chain aldehyde contents of the Kokuto.

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II. LITERATURE REVIEW

A. Sugarcane (Saccharum officinarum L.)

Sugarcane is an important crop due to the economic value of its products. Sugarcane is a tall thick perennial that belongs to the grass family (Poaceae). It has stout, jointed, fibrous stalk that are rich in sugar. The genus Saccharum comprises some different species, such as S. officinarum, S. barberi, S. sinense, S. edule, S. robustum and S. spontaneumi (Table 1). Saccharum officinarum and S. spontaneum are thought to be the ancestors of cultivated sugarcane. Saccharum officinarum was domesticated in Southeast Asia and originally derived from S. robustum. All of those species interbreed, and the major commercia

complexet al. 1987).

Table 1 Scientific classification of sugarcane

Kingdom Plantae

Division Magnoliophyta Class Liliopsida

Order Poales

Family Poaceae

Genus Saccharum L.

Species S. officinarum, S. arundinaceum, S. bengalense, S. edule, S. procerum, S. ravennae, S. Robustum

Sugarcane is a highly productive crop that has high photosynthetic ability as C4 plant. It requires strong sunlight and abundant water for

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months. Sugarcane stalk can grow to heights range 3.05–7.9 m and measuring 2.54–5.08 cm in diameter. Colors range from white, yellow, and green to purple.

Sugarcane is mainly cultivated in tropical and subtropical regions. Indonesia harvested about 436 847 ha and produced 77 MT/ha of cane for centrifugal sugar (Hadisaputro et al. 2008), of which almost three-quarters is on Java. Most of the remainder comes from Sumatra, Kalimantan and Sulawesi. The remainder is cultivated on sugar factory plantations, both in Java as well as on other islands where the dominant form of sugarcane cultivation is plantation-style. About 70 percents of the sugarcane areas are cultivated by farmers, mostly on small to medium sized holdings. FAO data (2005) mentioned Indonesia in rank 11th of sugarcane producer with 25 500 000 MT or equal to $ 529 635 000 (Table 2).

Table 2 Sugarcane producer countries in 2005

Rank Country Production ($1000) Production (MT)

1 Brazil 8 725 914 420 121 000

2 India 4 825 286 232 320 000

3 China 1 819 452 88 730 000

4 Thailand 1 029 610 49 572 000

5 Pakistan 981 260 47 244 100

6 Mexico 937 277 45 126 500

7 Colombia 827 669 39 849 240

8 Australia 794 369 38 246 000

9 Philippines 643 870 31 000 000

10 USA 535 948 25 803 960

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The sugarcane production areas in Japan are limited to the Southwestern islands, which are located between Taiwan and Kyushu Island, Japan. The cultivation areas of sugarcane are 20 970 ha in Okinawa Prefecture and 12 172 ha in Kagoshima Prefecture. Sugarcane occupies about 50% of agriculture area in Okinawa Prefecture and 60% in Tanegashima and Amami Oshima regions of Kagoshima Prefecture, and the crop is considered a key commodity in the region (Takagi et al. 2005).

Sugar is made by some plants to store energy that they do not need straight away, rather like animals make fat. Scientifically, sugar refers to any monosaccharide, also called simple sugar, i.e. glucose, fructose and galactose; or disaccharide, i.e. sucrose (saccharose), maltose and lactose (Belitz & Grosch 1999). In non-scientific use, the term sugar is used as a synonym for sucrose, also called table sugar, a white crystalline solid disaccharide. Many plants produce table sugar although only two are used commercially and these are commonly known as sugar-cane (Saccharum officinarum) and sugar-beet (Beta vulgaris). Manufacturing and preparing food may involve other sugars, including palm sugar and fructose, generally obtained from fruit.

Typical sugar content for mature cane would be 10% by weight but the figure depends on the variety and varies from season to season and location to location. Equally, the yield of cane from the field varies considerably but a rough and ready overall value to use in estimating sugar production is 100 MT of cane per hectare or 10 MT of sugar per hectare (Irei et al. 2005).

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B. Brown Sugar

Brown sugar is a sucrose sugar product with a distinctive brown color due to the presence of molasses resulted from caramelization of sucrose. It is made up of sucrose with greater amounts of ash, invert sugar and compounds derived from the process that give the sugar its characteristic flavor and color. Brown sugar is used in several confections such as caramels, toffees and butterscotch (Potter & Hotchkiss 1995). In Asia, Africa and South America non-centrifugal sugars are made for direct consumption and are known by a range of names: Kokuto in Japan, Gur in India and Bangladesh, Desi in Pakistan, Jaggery in Africa, and Panela in South America.

There are two categories of brown sugar: those produced directly from the cane juice at the place of origin and those that are produced during the refining of raw sugar. The first type includes a variety of molasses and syrups. The second type is coated brown or soft sugars and a variety of refinery molasses and golden syrups. The brown sugar types can be further divided into those where the crystals are separated (centrifuged) and those that are not separated (non-centrifuged) from the molasses.

Brown sugar is known to contain antioxidant compounds that have radical-scavenging activity and related functions such as anticancer effects and regulation of blood pressure. Takara et al.(2002, 2003) reported that Kokuto, Okinawan brown sugar (Figure 1) have antioxidative phenolic glycosides. They are expected to be effective in the prevention of many diseases and morbid states. More recently, Payet et al. (2005) investigated

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polyphenol content and volatile composition in seven cane brown sugars (four from La Réunion, two from Mauritius and one from France), and the relation to their unique free radical scavenging capacity.

Figure 1 Kokuto, Okinawan brown sugar.

C. Plant Wax

The term wax is derived from the Anglo-Saxon word weax which was used to describe the material in the honeycomb of bees. Wax generally refers to all waxlike solids and liquids found in nature and to those individual organic substances that crystallize on cooling and melt on heating. The nature of lipid constituents can vary greatly with the source of the waxy material, such as hydrocarbons, sterol esters, aliphatic aldehydes, primary and secondary alcohols, wax esters, diols, ketones, β-diketones, triacylglycerols, and so on (Dominguez & Heredia 1998).

The surfaces of plants are covered by several layers of lipophilic material, the outermost being the epicuticular waxes. They provide the

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hydrophobic barrier of the plant surface, and as such they function primarily to shed water and prevent nonstomatal water loss (Koch et al. 2006). In addition, they provide a first line of defense against bacterial and fungal pathogens and against abiotic stresses such as drought and UV damage (Reisige et al. 2006). Epicuticular waxes also play a role in plant-insect communication, by either attracting or deterring insects. This superficial material is synthesized by specialized cells in the outermost layers of the plant tissue. The amount of plant cuticular waxes produced is dependent on growth conditions, whilst chemical composition is less influenced by environmental factors. In general, wax yield from leaves and fruits of many species is ranging between 20 and 600 µ g/cm2

Sugarcane wax is the whitish to dark yellowish powdery deposit on the surface of the stalks of Saccharum officinarum L. During the milling of the cane, a large portion of this powdery substance is detached and mixed with the expressed juice. Sugarcane wax has been chemically defined as a complex and variable mixture of long-chain alkanes, hydrocarbons, fatty acids, ketones, aldehydes, alcohols, and esters (Nuissier et al. 2002; Purcell et al. 2005), and steroids such as β-sitosterol, stigmasterol, ketosteroids and hydroxyketosteroids (Goerges et al. 2006).

(Dominguez & Heredia 1998).

All compounds of plant wax have their own unique roles. However, only few researches have been investigated wax components functions in plant and their metabolism to date. Rutherford and Staden (1996) reported a correlation of high ratio of alcohol to aldehyde and shorter carbon chain length of sugarcane wax with stalk resistance to sugarcane borer Eldana

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saccharina Walker. Morris et al. (2000) described 1-octacosanal (C28-aldehyde) as the major component of wheat epicuticular wax that stimulates oviposition of the Hessian fly, Mayetiola destructor. Comparison of the activity of five straight-chain primary aldehydes with chain lengths from C22 to C30 revealed a relationship between chain length and the number of eggs laid by female Hessian flies, with 1-hexacosanal and 1-heptacosanal the most active of the aldehydes tested.

Wax is valuable source for many industries such as cosmetic, food ingredient, lubricant, printing and many other applications. Wax esters, oxo esters of long-chain fatty acids esterified with long-chain alcohols, can be used as high pressure lubricants, as replacements for hydraulic oil, and in the pharmaceuticals, leather, and food industries, as well as in candles and polishes. Long chain alkanals are known as favorable cosurfactants of liquids containing charged micelles (Meziani et al. 1997). Lately, long chain aliphatic alcohols of sugarcane wax have been used as cholesterol-lowering products (Taylor et al. 2000; Castano et al. 2003).

D. Wax Compositional Analysis

Wax content and composition, including long chain alcohols and aldehydes, can be analyzed with many techniques and instruments. The determination of wax composition often requires extensive sample extraction and preparation prior to instrumental analysis. Cuticular wax from potato leaf was extracted by dipping and shaking the leaves in dichloromethane (Szafranek & Synak 2006), as same as with cyclohexane in beeswax,

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spermaceti, carnauba, candellila and Japan waxes (Regert et al. 2005). Sorghum wax was extracted from grain sorghum using hot hexane and precipitating it in a -18°C (Hwang et al. 2002; Adhikari et al. 2006). Similar extraction concept was applied by Kanya et al. (2007), where sunflower seed wax was purified from oil refineries through extraction using solvents and precipitation with chilled acetone. Liquid-liquid extractions were successfully applied in beeswax, sugarcane and wheat (Irmak & Dunford 2005; Irmak et al. 2006).

Recently, non conventional methods for wax extraction have been developed with many techniques. Super critical carbon dioxide extractions were reported to have higher waxes yields than solvent extractions, i.e. cuticular wax from flax processing waste (Morrison et al. 2006), beeswax (Jackson & Eller 2006), and sugarcane crude wax (Lucas et al. 2007). Solvent-free extraction with high-intensity ultrasound treatment was studied in rice bran wax. Under sonochemical conditions bran wax could also be hydrolyzed yielding long chain alcohols (Cravotto et al. 2004). Molecular distillation was used to increase the purity of octacosanol (C28-alcohol) extracts from transesterified rice bran wax (Chen et al. 2005, 2007).

Compositional analysis of wax can be conducted with some instruments, whether qualitative or quantitative analysis. Thin layer chromatography was widely used to separate wax components (Hwang et al. 2002; Adhikari et al. 2006). Analytical methods of wax quantification were applied with high performance liquid chromatography (Hwang et al. 2002; 2005). Wax compounds were identified and confirmed by gas chromatography-mass spectrometry (Nuissier et al. 2002; Jiménez et al.

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2003; Kanya et al. 2007) and liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (Rezanka & Sigler 2006).

Main components of plant waxes were composed of fatty aldehydes, fatty alcohols, fatty acids, hydrocarbons, wax esters, sterol esters and triacylglycerols (Hwang et al. 2002; Adhikari et al. 2006), based on HPLC data. Recently, mixture of long chain aliphatic primary alcohols, policosanols, has been well investigated with gas chromatography technique (Cravotto et al. 2004; Irmak & Dunford 2005; Irmak et al. 2006; Wang et al. 2007). The mixture contains mainly docosanol (C22), tetracosanol (C24), hexacosanol (C26), octacosanol (C28), and triacontanol (C30), listed in Table 3.

Table 3 Mixture composition of aliphatic alcohols (policosanols)

No Structure Nomenclatur Molecular

Weight Carbon number

1 CH3(CH2)20CH2OH Docosanol 326.67 C22-alcohol 2 CH3(CH2)22CH2OH Tetracosanol 354.40 C24-alcohol 3 CH3(CH2)24CH2OH Hexacosanol 382.40 C26-alcohol 4 CH3(CH2)25CH2OH Heptacosanol 396.40 C27-alcohol 5 CH3(CH2)26CH2OH Octacosanol 410.74 C28-alcohol 6 CH3(CH2)27CH2OH Nonacosanol 424.74 C29-alcohol 7 CH3(CH2)28CH2OH Triacontanol 438.80 C30-alcohol

Sugarcane is the major source for the production of commercial policosanol products. Irmak et al. (2006) reported that sugarcane peel contained the highest amount of total policosanols, about 270 mg/kg. The total policosanol contents of sugarcane leaves (181 mg/kg) were quite similar to that of the wheat straw (164 mg/kg). Although, policosanol

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compositions of sugarcane plant parts varied significantly, C28 (about 81%) was the main component in all the sugarcane samples (Table 4). Policosanols in the perilla seeds were composed of 67–68 % octacosanol, 16–17% hexacosanol, and 6–9% triacontanol. The analysis of commercially milled wheat grain fractions, germ, bran, shorts, and flour showed that policosanols where concentrated in the bran. Wheat germ contained a significant amount of policosanols (10.1 mg/kg). These results were expected since policosanols are associated with lipids and wax in plant tissues. About 36% of the total policosanols in the wheat bran fraction was constituted of tetracosanol.

Table 4 Content and composition of policosanols in some materials No Material Policosanol content (mg/kg)

C24 C26 C28 C30

1 Whole sugarcane a 1.68 ± 0.08 0.9 ± 0.2 10 ± 0.2 1.0 ± 0.2

2 Sugarcane peel a 7.7 ± 0.2 2.3 ± 2 219 ± 3 16 ± 2

3 Sugarcane leaves a 29.4 ± 0.5 22.4 ± 0.8 84 ± 4 26.6 ± 0.6 4 Beeswax (brown) a 2.6 ± 0.1 1.7 ± 0.1 2.0 ± 0.1 5.7 ± 0.5 5 Beeswax (yellow) a 1.11 ± 0.06 0.86 ± 0.07 0.90 ± 0.03 2.3 ± 0.4 6 Perilla Seed (Korea) b 1.6 ± 0.13 17.6 ± 0.01 68.4 ± 1.44 6.8 ± 1.42 7 Perilla Seed (China) b 1.5 ± 0.06 _{16.6 ± 0.43} _{67.4 ± 0.07} _{9.1 ± 0.17}

8 Wheat germ c 1.4 ± 0.2 _n.d. _{2.9 ± 0.3} _{2.5 ± 0.3}

9 Wheat bran c 10.68 ± 0.01 _{4.87 ± 0.03} _{4.39 ± 0.02} _n.d.

10 Wheat shorts c 0.82 ± 0.01 _{0.45 ± 0.03} _{0.39 ± 0.01} _{0.22 ± 0.03}

Note: n.d. = not detected a

Irmak et al. (2006) b

Adhikari et al. (2006) c

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E. Policosanol in Human Health

Much research has been done on the potential of policosanols in lowering blood cholesterol and reducing the development of atherosclerotic plaques (Rodriguez et al. 1997; Arruzazabala et al. 2002). Studies have involved a wide range of subjects including experimental animals, healthy volunteers, and elderly patients with hypercholesterolemia. Several studies also have reported cardiovascular benefits of policosanols and its major component octacosanol, without major adverse effects.

Policosanols may decrease the risk of atheroma formation by reducing lipid levels, platelet aggregation, endothelial damage, and the development of foam cells. Octacosanol may decrease cholesterol synthesis in the liver before the generation of mevalonate. Octacosanol may down regulate the cellular expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Treatment with octacosanoic acid isolated and purified from sugarcane wax suppressed HMG-CoA reductase production in cultured fibroblasts, and this finding suggests a possible depression of de novo synthesis of the enzyme and cholesterol (Menendez et al. 2001). Furthermore, Singh et al. (2006) described that policosanol inhibits cholesterol synthesis in hepatoma cells by activation of AMP-kinase and is well established to suppress HMG-CoA reductase activity.

Research indicates that octacosanol at a dose of 5 mg/day may reduce LDL and total cholesterol in patients with borderline to mildly elevated serum triacylglycerol levels (Castano et al. 2003) and in normocholesterolemic and casein-induced hypercholesterolemic rabbits.

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Combination treatment policosanol (10 mg/day) and omega-3 fatty acid (1 g/day) has been associated with significant inhibition of platelet aggregation in rabbits compared with either drug alone (Castano et al. 2006).

Absorption and metabolism of nonesterified policosanol have been studied by many researchers. Hargrove et al. (2003) mentioned that the liver may be converting octacosanol to long chain fatty acids, which were subsequently taken up by muscle. The study identified that the administered octacosanol was found in sterols and triacylglycerol, which indicated conversion to fatty acids and esterification. Policosanol metabolism is linked to fatty acid metabolism via β-oxidation. Menendez et al. (2005) demonstrated that octacosanoic acid was formed after incubation of fibroblast cultures with 3H-octacosanol and after oral dosing with policosanol to rats. In addition, shortened saturated (myristic, palmitic and stearic) and unsaturated (oleic, palmitoleic) fatty acid were also formed after oral dosing with policosanol to monkeys.

More rapid onset of effects suggests that oxidation of policosanols to very long-chain fatty acids may be necessary for their hypocholesterolemic actions and enhance the breakdown of LDL particles (McCarty 2005). In another study, dietary octacosanol was thought to increase lipid catabolism to generate more energy for improvement of motor endurance; and this action may contribute in reductions of plasma triglyceride levels. Octacosanol is already recognized as an ergogenic product used to enhance athletic performance (Taylor et al. 2003).

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In an effort to identify the potential mechanism for the antiatherogenic effects of policosanols, Ng et al. (2005) investigated the effects of these natural compounds on LDL oxidation and bile acid excretion. Policosanols showed no antioxidant activity in human LDL particles but increased bile acid secretion in hamsters.

However, other research groups using policosanol from alternative sources have failed to reproduce the efficacy of these alcohols observed in earlier studies. Lin et al. (2004) reported that wheat germ policosanol 20 mg/day had no beneficial effects on blood lipid profiles in subjects with normal to mildly elevated cholesterol concentrations. Kassis et al. (2007) mentioned that sugarcane policosanol treatment at a dose of 275 mg/kg diet had any significant cholesterol-lowering effect on plasma lipid levels in Golden Syrian hamsters.

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III. MATERIALS AND METHODS

A. Materials 1. Samples

Whole parts of sugarcane stalk of Ni 15 cultivar, rind parts of seven varieties of sugarcane (Ni 13, Ni 17, Ni 22, NiF 8, NCo 310, F 161 and F 177 cultivars), seven types of Kokuto (type A–G) and two non-Japan brown sugars (type P from Thailand and type Q from Bolivia) were used as materials. Rind and pith parts of sugarcane stalk (Figure 2) were separated by cane separator, known as CSS (Cane Separation System), from Mitsubishi Engineering Co. Ltd., Japan (Figure 3) and by hand peeling. Kokuto samples were available products from 2007 to 2008 production year of Kokuto manufacturers in Okinawa Prefecture, Japan. Cane juice and end product from Kokuto production line were also used in this study.

Figure 2 Rind (a) and (b) pith parts of sugarcane separated by CSS.

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Figure 3 Cane Separation System (CSS).

2. Materials and Reagents

The policosanol standards used consist of docosanol (C22), tetracosanol (C24), hexacosanol (C26), octacosanol (C28), and triacontanol (C30). They were purchased from Sigma (Sigma Chemical, St. Louis, MO). Derivatization reagent N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was purchased from GL Science (Japan). Pyridinium chlorochromate, from Sigma (Sigma Chemical, St. Louis, MO), was used for synthesis of long chain aldehyde standards.

Several chemicals were used as standards in analysis of wax composition. They were triacontane, octacosanol, cholesteryl oleate, lignoceric acid, methyl palmitate and stigmasterol from Sigma (Sigma Chemical, St. Louis, MO), synthesized aldehyde (octacosanal), and triolein (Nakarai Chemicals Ltd., Japan). HPLC grade hexane (Wako Pure Chemical Industries Ltd., Japan) and methyl tert-butyl ether (Kanto Chemicals Co. Inc., Japan) were used as mobile phases in HPLC analysis.

All reagents and chemicals were of analytical and HPLC grade, unless specified.

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B. Methods

Samples (sugarcane and Kokuto) were prepared and kept dry before use. Sugarcane and cane juice samples were freeze-dried for 24 h. Then all samples were crushed and ground with dry blender. Parts of sugarcane samples were separated by CSS and hand peeling. Those sugarcane samples were extracted for their wax, policosanol and long chain aldehyde compounds, and analyzed as shown in experimental design below (Figure 4). Wax composition of extracted samples were qualitatively separated by thin-layer chromatography (TLC) and quantified by HPLC-ELSD. Policosanol and long chain aldehyde contents and compositions were analyzed with GC-FID and its compounds were identified with GC-MS. All listed quantification runs were analyzed statistically ANOVA with completely randomized design.

Figure 4 Experimental designs of sugarcane wax, policosanol and long chain aldehyde analysis.

Sugarcane

CSS separation

Ni 15 cultivar

Whole, pith and rind

Policosanol and long chain aldehyde

analysis

Rind

Extration times variation

Policosanol and long chain aldehyde

analysis

Rinds of 8 cultivars

Policosanol and long chain aldehyde

analysis Hand peeling separation (rinds) Mature cane (12 months) Wax compositional analysis Harvesting times (9 and 12 months)

Policosanol and long chain aldehyde

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Kokuto samples were obtained from two types Kokuto factories (Figure 5); there were the traditional factory with open pan method (Kokuto A) and the modern factory with vacuum pan technology (Kokuto Non-A). Raw cane juice, clear juice and end product of both production lines were compared to investigate the effect of Kokuto production types on policosanol and long chain aldehyde contents. Several extraction methods and times were tried as shown in the experimental design (Figure 6). Samples of Kokuto B–G were collected from the modern factories (Kokuto Non-A type). Policosanol and long chain aldehyde contents and compositions were analyzed with GC-FID and its compounds were identified with GC-MS. All listed runs were analyzed statistically ANOVA with completely randomized design.

Production Line Factory Non-A

Milling

Open pans evaporation

Kokuto

Production Line Factory A Raw cane juice

Cane

Coagulation

Clear juice

Concentrated sugar syrup

Milling

Vacuum pans evaporation

Kokuto

Raw cane juice

Cane

Coagulation

Concentrated sugar syrup

Clarification

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Figure 6 Experimental designs of policosanol and long chain aldehyde analysis in Kokuto.

1. Wax compositional analysis by TLC and HPLC-ELSD a. Standard preparation

Several compounds were used as standards of wax components: triacontane (for hydrocarbon), synthesized octacosanal (aldehyde), cholesteryl oleate (sterol ester), octacosanol (alcohol), triolein (triacylglycerol), lignoceric acid (acid), methyl palmitate (methyl ester) and stigma sterol (sterol). Those standards were prepared in toluene for TLC analysis and in chloroform with different concentrations (0.05–0.50 mg/ml) covering the levels of sample components for quantification of each wax component by HPLC analysis.

Kokuto

Factory A

Kokuto A

Extraction methods variation Policosanol and long chain aldehyde analysis Extration times variation Policosanol and long chain aldehyde analysis

Cane juice and Kokuto A

Policosanol and long chain aldehyde analysis

Factory Non-A

Cane & clear juices and KokutoNon-A Policosanol and long chain aldehyde analysis KokutoB-G Policosanol and long chain aldehyde analysis

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b.Sugarcane wax extraction

Briefly, 10 g of freeze-dried rind of sugarcane were placed in Advantec No. 84 thimble filter and extracted using soxhlet for 4 h with about 150 ml of hexane, methanol (20:1 v/v). The solvent solution was removed from the extract with rotary-evaporator under vacuum condition at 40°C. The amount of dry wax extract was weighed and diluted with toluene for TLC analysis and with chloroform down to 1–2 mg/ml to the reach detection level of ELSD used in HPLC analysis.

c. TLC Analysis

Thin-layer chromatography (TLC) of waxy materials extracted from sugarcane rind samples using a silica gel 60, 20 cm × 10 cm × 250 µm TLC plate. Cholesteryl oleate, methyl palmitate, synthesized octacosanal, triolein, lignoceric acid, octacosanol and stigma sterol were used as standards. Samples and standards were eluted with two steps of developing solvents. The first solvent mixture, comprising hexane, diethyl ether, acetic acid (95:5:1 v/v/v), was allowed to travel 10 cm before the plate was removed and the solvent allowed to evaporate. Once dried, the plate was redeveloped in hexane, diethyl ether, acetic acid (80:20:1 v/v/v) to the top of the plate. Developed bands were visualized by spraying the plate with 10% cupric sulfate solution containing 8% phosphoric acid. Then the TLC plate was dried in heater until the developed bands were charred.

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d.HPLC-ELSD analysis

Wax components were separated and determined as previously reported by Adhikari et al. (2006), using HPLC equipped with a Luna silica column (250 mm × 4.6 mm i.d. × 5 µm film thickness) connected to a guard column (4 × 3 mm i.d.) supplied by Phenomenex (Torrance, CA). Two Shimadzu LC-10AD-VP pumps were operated in combination with a Shimadzu SCL-10A-VP gradient controller. The column and guard column temperature were kept constant at 40°C by a Shimadzu CTO-10AC-VP. Shimadzu model LT evaporative light scattering detector was operated at 50°C with nitrogen pressure 350 kPa. Mobile phases consisted of a gradient of hexane (solvent A) and methyl tert-butyl ether containing 0.2% acetic acid (solvent B), with the following profile: 0–2 min, 100% A; 3–10 min, 95% A; 14 min, 55% A; 23–26 min, 0% A; then 27–40 min, 100% A. Flow rate of mobile phase was 1 ml/min. Injection volume of samples and standard were 5 µl. The detection limit of this analysis was 0.01 mg/ml.

Several compounds were used as standards, i.e. triacontane, cholesteryl oleate, synthesized octacosanal, triolein, lignoceric acid, octacosanol and stigma sterol. Wax component contents in analyzed samples were determined based on the relation between peak area and calibration curve of each standards. Wax contents were calculated in percentage (%, w/w) and concentration (mg/100 g sample, wet weight basis) by following equations:

Component A (%) =Peak area of component A

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Concentration (mg/100g) =

Content�mg_ml�∗× Volume of extract (ml) × 100 Sample weight (g)

where content (*) was calculated by calibration curve of its standard.

2. Policosanol and long chain aldehyde analysis by GC-FID a. Standard preparation

Mixed policosanol standards for policosanol compounds quantification were prepared in toluene. Aldehyde standards were synthesized from their alcohols form by oxidation with pyridinium chlorochromate, as described by Pérez-Camino et al. (2003). Each 1 mM corresponding alcohols standards (19.14 mg hexacosanol, 20.54 mg octacosanol, 21.94 mg triacontanol) and 9 mM pyridinium chlorochromate (97.5 mg) were stirred in 50 ml dichloromethane for 1.5 h at room temperature. The reaction mixture was eluted with dichloromethane through a short column (6 × 2 cm I.D.), packed with silica gel 60. Then the reaction product was dried by N2 gas and diluted

in toluene. The synthesized long chain aldehyde standards were checked with GC-FID analysis.

b.Sample extraction

Kokuto A samples were extracted with two extraction methods in order to investigate the optimum extraction method; they were liquid-liquid extraction (LLE) by the method of Irmak et al. (2006) and solid-liquid extraction (SLE, soxhlet method). The optimum extraction

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method was applied to policosanol and long chain aldehyde contents analysis in sugarcane and other Kokuto samples.

Liquid-liquid extraction (LLE) method. Briefly, 6 g of Kokuto sample was mixed with 50 ml of 1.0 N NaOH and 50 ml of methanol and subsequently hydrolyzed by refluxing in a heater for 30 min. After cooling, the mixture was filtered through Advantec No. 5A filter paper under vacuum condition. Then Millipore water was added to the filtrate. The solution was extracted three times with 50 ml diethyl ether. Combined diethyl ether phases from three extractions were neutralized with Millipore water until pH of water phase reached 7. The extract was dried over 50 g of anhydrous sodium sulfate by storing at 4°C for one night. Then the solvent was removed from the extract with rotary-evaporator under vacuum condition. The dry extract was diluted in toluene or chloroform and made up to 2 ml prior to analysis.

Solid-liquid extraction (SLE) method. Briefly, 10 g of freeze-dried sugarcane or 6 g of Kokuto were placed in Advantec No. 84 thimble filter and extracted using soxhlet with about 150 ml of several systems of organic solvent. The solvent systems were Soxhlet A (chloroform, methanol (2:1 v/v)); Soxhlet B (hexane, methanol (10:1 v/v)); Soxhlet C (hexane, methanol (20:1 v/v)); Soxhlet D (hexane, methanol (30:1 v/v)); and Soxhlet E (hexane). Several extraction times were also attempted in order to investigate the heat stability and optimum extraction condition of policosanol and long chain aldehyde compounds in sugarcane and Kokuto samples. The solvent solution was removed from the extract with rotary-evaporator under vacuum condition at 40°C.

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Then the dry extract was diluted in toluene and made up to 2 ml for GC-FID analysis, whereas for GC-MS analysis, the extract was diluted in chloroform.

c. Gas chromatography analysis (GC-FID)

Shimadzu GC 17-A equipped with a fused capillary column (DB 5, 0.25 mm i.d. × 30 m × 0.25 µm film thickness) from J&W Scientific (Folsom, CA) and a flame ionization detector were used for policosanol and long chain aldehyde quantitative analysis. The GC injector was set at 350°C and the flame ionized detector 350°C. The samples (1 µl) were injected with split ratio of 1:10. The carrier gas was helium with a flow rate of 1 ml/min. The oven temperature was programmed at 150°C as initial temperature, raised to 320°C at 4°C/min and maintained at 320°C for 15 min.

By injecting mixture standards of policosanol and aldehyde with different concentrations covering the levels of sample extracts, relation between concentration and peak height was plotted for calibration. Concentrations of standard solutions for the standard curve were 0.05–0.50 mg/ml. The detection limit of this analysis was 0.01 mg/ml. Policosanol and long chain aldehyde concentrations were calculated as mg/100 g sample (wet weight basis for sugarcane) by this following equation:

Concentration (mg/100g) =

Content�mg ml�

∗

× Volume of extract (ml) × 100 Sample weight (g)

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3. Policosanol and long chain aldehyde identification by GC-MS

The silylated derivatization was used for mass spectrum analysis of policosanol, however mass spectrum of aldehyde was analyzed without silylation. In this case, policosanol was identified as its trimethylsilyl derivates. N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was used as silylation reagent. The derivatization solution was made by mixing 0.5 ml of sample in chloroform with 250 µl MSTFA. The solution was heated at 50°C for 15 min. The volume of the mixed solution was made up to 1 ml with chloroform for GC-MS analysis. Derivatization was also applied for policosanol standard solution at concentration of 0.1 mg/ml.

The analysis was performed using Shimadzu GC-MS QP-2010 with a fused capillary column DB-5 MS (0.25 mm i.d. × 30 m × 0.25 µm film thickness) from J&W Scientific (Folsom, CA) under the same GC conditions. The samples (0.3 µl) were injected with split ratio of 1:10. For MS detection, the electron impact (EI) ion source and transfer line temperature were set at 200°C and 280°C. The ionization energy was mode at 70 eV. The mass acquisition scan range and rate were 30–500 amu and 2 scans/s. Identification of each policosanol or aldehyde was conducted by comparing its retention time and mass spectrum directly to those of its respective standard. The mass spectrums were also confirmed with NIST 2005 Mass Spectral Library by GCMSsolution software (Shimadzu).

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4. Statistical analysis

All extraction runs and analysis were carried out in triplicate and analyzed statistically using ANOVA with completely randomized design. The mean values of analysis results were reported. The data were analyzed by SPSS Version 13.0 for Windows (SPSS Inc., USA, 2003). Univariate comparisons of the various means were carried out by post hoc test of Duncan at p = 0.05. Nine experimental designs were attempted with statistical analysis, there were:

a. The effect of rind parts of sugarcane cultivars on wax composition (3 samples, i.e. NiF 8, Ni 15 and Ni 22 cultivars).

b. The effect of different parts of sugarcane of Ni 15 cultivar on the policosanol and long chain aldehyde contents (3 samples, i.e. rind, pith and whole stalk).

c. The effect of extraction times of rind part of Ni 15 cultivar on the policosanol and long chain aldehyde contents (5 extraction times, i.e. 2, 4, 8, 16 and 24 h).

d. The effect of sugarcane cultivars on the policosanol and long chain aldehyde contents of the sugarcane rinds (7 samples, i.e. Ni 13, Ni 15, Ni 17, NiF 8, NCo 310, F 161 and F 177 cultivars).

e. The effect of sugarcane harvesting time on the increasing of total policosanol and long chain aldehyde contents of the sugarcane rinds (5 samples, i.e. NCo 310, Ni 15, Ni 17, NiF 8 and Ni 22).

f. The effect of extraction methods of Kokuto A on the policosanol and long chain aldehyde contents (6 extraction methods, i.e. a liquid-liquid extraction method and 5 soxhlet extraction methods).

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g. The effect of extraction times of Kokuto A on the policosanol and long chain aldehyde contents (5 extraction times, i.e. 4, 8, 16, 24 and 32 h). h. The effect of Kokuto types on the policosanol and long chain aldehyde

contents of the Kokuto (9 samples, i.e. type A–G, P and Q).

i. The effect of Kokuto production types on the policosanol and long chain aldehyde contents (5 samples, i.e. raw juice and end product of Kokuto factory A; and raw juice, clear juice and end product of Kokuto factory Non-A).

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IV. RESULTS AND DISCUSSION

A. Sugarcane Wax Composition

The qualitative separation of most of the major classes of aliphatic wax components could be achieved by silica gel of thin layer chromatography (Figure 7). Compositions of the waxy materials from sugarcane cultivars were all similar. They were composed of sterol, alcohol, acid, triacylglycerol, methyl ester, aldehyde and sterol ester. Thin layer chromatography had been widely used for qualitative analysis in waxy materials (Hwang et al. 2002; Adhikari et al. 2006; Webster et al. 2006).

Figure 7 Thin layer chromatography of waxes of several sugarcane cultivars. S: Standard.

S NiF 8 Ni 22 Ni 15

Plant sterol Policosanol Acid Triacylglycerol Sterol ester

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Wax components of sugarcane on TLC plate could finally be visualized by cupric sulfate charring. Each component of waxy materials was well charred and separated, except for sterol ester which split into two smear bands. These two separated bands were identified by Adhikari et al. (2006) as wax ester and sterol ester. Besides this, methyl ester and aldehyde were appeared as a single spot, while acid and policosanol as closed dense spots.

Separation of cane wax components by high performance liquid chromatography (HPLC) were further quantified with evaporative light-scattering detector (ELSD). Five peaks in HPLC chromatogram were identified as groups of aldehyde, sterol ester, triacylglycerol, acid, alcohol, and sterol (Figure 8). Wax component contents in analyzed samples were determined based on the relation between peak area and calibration curve of each standards. An example of standard curve of triacylglycerol is shown in Figure 9, it has a linear equation of y = 1E+07x – 52429. These standards (Figure 10) had different concentrations covering the levels of components in samples, i.e. 0.05–0.50 mg/ml.

This result confirmed the same result obtained by TLC. Every peak represented one group of wax components, except for peak 1 which was a mixture of aldehyde and sterol ester. However, Hwang et al. (2002) and Adhikari et al. (2006) identified this peak as mixture of aldehyde, sterol ester and also wax ester. A very weak respond of hydrocarbon compound somewhat was detected (after 3 minutes retention), but it was not enough for making a single peak.

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Figure 8 HPLC chromatogram of sugarcane rind of Ni 15 cultivar. The chromatogram was obtained by HPLC-ELSD method.

Peak 1 : Aldehyde, sterol ester Peak 4 : Alcohol Peak 2 : Triacylglycerol Peak 5 : Sterol Peak 3 : Acid

Figure 9 Standard curve of triacylglycerol.

y = 1E+07x - 524294 R² = 0.9987

0 1,000,000 2,000,000 3,000,000 4,000,000 5,000,000 6,000,000

0.00 0.10 0.20 0.30 0.40 0.50 0.60

rea

Concentration (mg/ml)

0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.2 0.4 0.6 0.8 1.0

0.0

2 3 4

Time (min) V

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Figure 10 HLPC chromatograms of wax composition standards. (a) aldehyde, (b) sterol ester, (c) triacylglycerol, (d) acid, (e) alcohol, (f) sterol. Standards concentrations were 0.5 mg/ml (a-d) and 0.4 mg/ml (e-f). The chromatograms were obtained by HPLC-ELSD method.

(a) (b)

(c) (d)

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Wax components in sugarcane rind were composed of mixture of aldehydes and sterol esters (55.1–60.4%), alcohols (31.8–39.8%), triacylglycerols (2.3–4.6%), acids (2.0–2.7%), and sterols (0.5–0.9%), respectively (Table 5, Figure 8). Significant differences (p < 0.05) were observed in all wax composition of Okinawan sugarcane cultivars. The highest content of wax component was found in sugarcane rind of Ni 22 cultivar with about 1 g of mixture of aldehydes and sterol esters per 100 g sample (wet weight basis). Followed with mixture of aldehydes and sterol esters of NiF 8 and Ni 15 cultivars, i.e. 751.0 and 594.6 mg/100 g. The alcohol group, policosanols, of Ni 22 cultivar sample was 462.9 mg/100 g; while sterol group was less abundant and its response became too small for making a detectable peak.

Table 5 Wax compositions of rind part of Okinawan sugarcane cultivars, obtained by HPLC-ELSD.

Cultivar Aldehyde,

sterol ester* Triacylgycerol Acid Alcohol Sterol

% (w/w)

NiF 8 55.1 ± 1.1 b 3.7 ± 0.4 b 2.7 ± 0.1 a 39.8 ± 4.0 a 0.5 ± 0.03 Ni 15

60.5 ± 3.0 a 4.6 ± 0.6 a 2.4 ± 0.4 ab 31.8 ± 2.1 b 0.9 ± 0.2 Ni 22

58.5 ± 1.8 ab 2.3 ± 0.2 c 2.0 ± 0.2 b 37.3 ± 1.9 ab n.d.

mg/100 g

‡_c

NiF 8 751.0 ± 29.4 q 101.7 ± 10.7 p 62.0 ± 1.6 p 367.5 ± 8.1 q 33.3 ± 0.01 Ni 15

594.6 ± 32.6 r 80.2 ± 6.0 q 39.0 ± 1.4 q 216.4 ± 14.7 r 25.3 ± 0.2 Ni 22

1027.1 ± 66.5 p 97.0 ± 6.9 p 63.2 ± 3.1 p 462.9 ± 7.6 p n.d.r

*Calculated as aldehyde group. ‡

Not detected. a

Data are means ±S.D. (n = 3). Means in the same column with the same letter are not significantly different (p > 0.05).

(62)

Similar separation method and stationary phase of wax component of plant waxes were applied in grain sorghum (Hwang et al. 2002), perilla and sesame seeds (Adhikari et al. 2006), potato leaves (Szafranek & Synak 2006), and calanoid copepod Calanus finmarchicus (Webster et al. 2006). The amounts and compositions of these waxes were markedly different depended on their genetics, bio-functions, plant growth conditions, and environments. Another HPLC technique of wax components separation was using alumina as stationary phase (Nordback & Lundberg 1999).

Plant waxes are a complex heterogeneous mixture of very long chain (C20–C34) fatty acids and their derivatives. During wax biosynthesis very long chain fatty acids are further modified to aldehydes, alkanes, ketones, and so on. Grain sorghum and carnauba waxes compositions were well investigated by Hwang et al. (2002). Grain shorghum wax was composed of 46.3% (w/w) fatty aldehydes, 7.5% fatty acids, 41.0% fatty alcohols, 0.7% hydrocarbons, 1.4% wax esters and sterol esters, and 0.9% triacylglycerols. Carnauba wax contained of 34.3% wax esters, 5.1% fatty acids, undetermined amount of fatty alcohols, and 3.0% triacylglycerols, determined by HPLC.

Major components of the waxy materials from Korean and Chinese perilla seeds were alcohols (25.5 and 34.8%), hydrocarbons (18.8 and 10.5%), wax esters, steryl esters and aldehydes (53.0 and 49.8%), acids (1.7 and 2.1%), and triacylglycerols (1.0 and 2.9%), based on HPLC data were reported by Adhikari et al. (2006). The principal components of leaf cuticular waxes from potato varieties were very long chain n-alkanes, 2-methylalkanes and 3-methylalkanes (3.1–4.6 µg cm-2), primary alcohols (0.3–0.7 µg cm-2), fatty acids (0.3–0.6 µg cm-2), and wax esters (0.1–0.4 µg

(63)

cm-2

In general, sugarcane stalk was composed of sugars 12–16%, water 70–74%, pith fiber 7%, rind fiber 7%, and epidermis 0.1%. Wax would abundantly found in rind part as epicuticular wax. Figure 11 shows the comparison of policosanol and long chain aldehyde contents as wax components in sugarcane stalk. Rind and pith samples were separated by CSS (Cane Separation System). This study discovered that policosanol and long chain aldehyde compositions of sugarcane parts varied significantly. Policosanol and aldehyde compounds in the rind part of sugarcane were found much abundant than in pith.

), analyzed by GC-FID (Szafranek & Synak 2006). Methyl ketones, sterols, β-amyrin, benzoic acid esters and fatty acid methyl, ethyl, isopropyl and phenylethyl esters were found in potato waxes. A new group of cuticular wax constituents consisting of free 2-alkanols with odd and even numbers of carbon atoms ranging from C25 to C30 was also identified.

Figure 11 Policosanol and long chain aldehyde contents in sugarcane Ni 15 cultivar, obtained by GC-FID analysis.

Means in the same group with the same letter are not significantly different (p > 0.05).

0 20 40 60 80 100

Rind Pith Whole stalk

A m ou n t ( m g/ 100 g) Policosanol Aldehyde a p

c _r

b q

(64)

The whole stalk of sugarcane Ni 15 cultivar contained 35 mg policosanols and 24 mg aldehydes per 100 g of wet weight basis. The policosanol and aldehyde contents in pith part were negligible (about 1 mg/100 g), however, policosanols and adehydes of rind part was found in high concentration, i.e. 80 mg/100 g. This result is associated with surface wax present in rind of cane. The surface waxes protect plants from water lost and environmental stress (Koch et al. 2006; Dominguez & Herdia 1996). Separation with CSS would make loosing an amount of epicuticular waxes in rind, while either it separated to epidermis wax chamber or attached in rolls and blades of CSS. In this way the hand peeled rind samples were also investigated.

Rutherford and Staden (1996) reported a suggestion of sugarcane surface wax component towards resistance to borer Eldana saccharina Walker. In their investigation, a high ratio of alcohol to aldehyde and shorter chain length appeared to be associated with cane resistance. Furthermore, Purcell et al. (2005) described epicuticular wax as a potential genetic marker and predictor of desirable plant traits. According to that a comprehensive study of wax compositional analysis and eco-geochemistry of Okinawan sugarcane cultivars is needed.

(65)

B. GC Chromatogram and Mass Spectrum of Policosanols and Long Chain Aldehydes

Figure 12 shows the typical chromatogram of standard mixture and sample extracts. All compounds of standard mixture were completely separated to every single peak (Figure 12a). The retention time of three synthesized aldehyde compounds (hexacosanal, octacosanal, and triacontanal) were detected 1 minute faster than their corresponding alcohol compounds. Quantification of each policosanol and aldehyde compounds in extracted samples was determined base on the retention time and peak height of each referred standard.

Derivatization is primarily performed to modify an analyte’s functionality in order to enable chromatographic separations. The formation of chemical derivatives to facilitate meaningful analysis has long been a common practice in gas chromatography. The use of MSTFA as silylation reagent for derivatization has been reported in policosanol determination using GC-FID by some researchers (Adhikari et al. 2006; Morrison et al. 2006), however, the use of MSTFA in the pre-study was found in less effective for policosanol determination comparing to direct analysis. Besides this, the use of internal standard (i.e. octacosanoic acid) was avoided due to a number of closed peaks in interest area of chromatogram of samples (Figure 12b and 12c).

Policosanol and long chain aldehyde contents in analyzed samples were determined based on the relation between peak height and calibration curve of each standards. An example of standard curve of octacosanol is shown in Figure 13, it has a linear equation of y = 24006x + 173.2.

(66)

Figure 12 Typical gas chromatogram of (a) policosanol and long chain aldehyde standards, (b) Kokuto A, (c) sugarcane rind of Ni 15 cultivar. The chromatograms were obtained by GC-FID.

Peak 1 : C22-OH Peak 2 : C24-OH

Peak 3 : C26-OH Peak 3a Peak 4 : C28-OH Peak 4

: C26-CHO

Peak 5 : C30-OH Peak 5

: C28-CHO

: C30-CHO

0.0 10.0 20.0 30.0 40.0 50.0 (min)

(b) 4 5a 5 3 3a 2 1 4a

0.0 10.0 20.0 30.0 40.0

4a 4 5a 5 (min) 3 3a 2 1 (a)

0.0 10.0 20.0 30.0 40.0 50.0 (min)

(c)

4 5a

5 3 3a 2 1 4a

(1)

1 (g) Mass spectrum of trimethylsilyl ether of C30 of standard

1 (h) Mass spectrum of trimethylsilyl ether of C30 of sample

50 100 150 200 250 300 350 400 450

0 10 20 30 40 50 60 70 80 90 100 110 120

495

75 57 43

496 71

55 ₈₃97 103

111

32 125 ₂₀₈ ₂₈₁

m/z

50 100 150 200 250 300 350 400 450

0 10 20 30 40 50 60 70 80 90 100 110 120

495

75 57 43

496 71₈₃

55 103

111 125 39

355 32

m/z

% %

(2)

Appendix 2Mass spectrum of long chain aldehydes.

2 (a) Mass spectrum of C26 aldehyde of standard

2 (b) Mass spectrum of C26 aldehyde of sample

2 (c) Mass spectrum of C30 aldehyde of standard

2 (d) Mass spectrum of C30 aldehyde of sample

305 310 315 320 325 330 335 340 345 350 355 360 365 370 375. 380 0.0

2.5 5.0 %

362

334 306

320

380 336

m/z

280 290 300 310 320 330 340 350 360

0.0 5.0 10.0 %

362

334 306

292 320

278

m/z

350 360 370 380 390 400 410 420 430 440

0 10 %

418

390 362

355 429

348

376 436

m/z

290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 0.0

5.0 10.0 %

418

390 362

355 334

292 306 320 348 376 391 429 436

(3)

Appendix 3 Statistical analysis of total policosanol content in sugarcane rind, obtained by SPSS Version 13.0 for Windows.

Univariate Analysis of Variance

Between-Subjects Factors

Ni 13 3

Ni 15 3

Ni 17 3

NiF 8 3

NCo 310 3

F 161 3

F 177 3

1.00 2.00 3.00 4.00 5.00 6.00 7.00 Factor

Value Label N

De scriptive S tatistics

Dependent Variable: Respond

71.1667 4.37705 3

79.3933 6.97768 3

123.6467 7.21361 3

88.4233 7.34034 3

59.6833 4.62986 3

68.6900 6.20096 3

85.0767 6.31412 3

82.2971 20.36999 21

Factor Ni 13 Ni 15 Ni 17 NiF 8 NCo 310 F 161 F 177 Total

Mean St d. Deviation N

Le vene's Test of Equa lity of Error Va riancesa

Dependent Variable: Respond

.354 6 14 .896

F df1 df2 Sig.

Tests the null hypothes is that t he error variance o the dependent variable is equal across groups.

Design: Int ercept+Fact or a.

Te sts of Betw een-Subje cts Effe cts

Dependent Variable: Respond

7751.692a 6 1291.949 33.064 .000 142229.214 1 142229.214 3639.983 .000 7751.692 6 1291.949 33.064 .000

547.038 14 39.074 150527.944 21

8298.730 20 Source

Correc ted Model Int ercept

Factor Error Total

Correc ted Total

Ty pe III Sum

of Squares df Mean Square F Sig.

R Squared = . 934 (Adjusted R Squared = .906) a.

(4)

Estimated Marginal Means

1. Grand Mean

Dependent Variable: Res pond

82.297 1.364 79.372 85.223

Mean Std. Error Lower Bound Upper Bound 95% Confidence Interval

2. Factor

Dependent Variable: Respond

71.167 3.609 63.426 78.907 79.393 3.609 71.653 87.134 123.647 3.609 115.906 131.387 88.423 3.609 80.683 96.164 59.683 3.609 51.943 67.424 68.690 3.609 60.950 76.430 85.077 3.609 77.336 92.817 Factor

Ni 13 Ni 15 Ni 17 NiF 8 NCo 310 F 161 F 177

Mean St d. E rror Lower Bound Upper Bound 95% Confidenc e Int erval

Post Hoc Tests Factor

Homogeneous Subsets

Respond

Duncana,b

3 59.6833

3 68.6900 68.6900 3 71.1667

3 79.3933 79.3933

3 85.0767

3 88.4233

3 123.6467

.099 .065 .114 1.000 Factor

NCo 310 F 161 Ni 13 Ni 15 F 177 NiF 8 Ni 17 Sig.

N 1 2 3 4

Subset

Means for groups in hom ogeneous s ubs ets are displayed. Based on Type III Sum of Squares

The error term is Mean Square(Error) = 39.074. Us es Harm onic Mean Sample Size = 3.000. a.

Alpha = .05. b.

(5)

Sugarcane contained many waxy materials, including policosanols and long chain aldehydes. Sugarcane wax components could be separated and analyzed with thin layer chromatography, high performance liquid chromatography and gas chromatography techniques. This study gives information of functional compounds potentially contained in sugarcane and its product Kokuto, an Okinawan brown sugar.

The study showed a simple and effective extraction method for policosanols and long chain aldehydes in sugarcane and Kokuto, those were solid liquid extraction with a mixture of hexane, methanol (20:1 v/v) for policosanol; and a mixture of chloroform, methanol (2:1 v/v) for long chain aldehyde. Octacosanol and octacosanal were the major component in the whole extracts, analyzed by GC-FID and GC-MS. Sugarcane rinds contained 53–60% aldehydes, sterol esters and wax esters, 32–40% alcohols, 2–4% triacylglycerols, 2–3% acids, and 0.6–0.9% sterols, analyzed by HPLC-ELSD. The wax contents and compositions of the samples analyzed varied significantly with the sugarcane cultivars.

This study discovered that policosanol and long chain aldehyde compositions of sugarcane parts varied significantly, as same as, with its contents in the rind parts of sugarcane cultivars. Hand peeled cane rinds contained wax components higher than in cane piths, i.e. cq. 500 mg policosanols and 600 g aldehydes per 100 g sample (Ni 22 cultivar). The amount of sugarcane surface wax produced is dependent on the varieties and growth condition. Increasing of aldehyde compounds were found higher than alcohol compounds.

(6)

Policosanol and long chain aldehyde could also be found in Kokuto. Their contents corresponded to their production system. Those in Kokuto A could reach 85 mg policosanols and 8 mg aldehydes per 100 g sample, whereas

Kokuto Non-A 8–12 mg policosanols per 100 g sample and negligible amount of aldehydes. The policosanol and long chain aldehyde compositions of the samples analyzed varied significantly with Kokuto types.

Waxes, policosanols and aldehydes in sugarcane (Saccharum officinarum L.) and okinawan brown sugar (kokuto)

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ABSTRACT

RINGKASAN

PREFACE

DECLARATION OF ORIGINALITY

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RINGKASAN

PREFACE

CURRICULUM VITAE

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF APPENDIX

LIST OF ABBREVIATIONS

I. INTRODUCTION

II. LITERATURE REVIEW

III. MATERIALS AND METHODS

IV. RESULTS AND DISCUSSION

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Waxes, policosanols and aldehydes in sugarcane (Saccharum officinarum L.) and okinawan brown sugar (kokuto)

DECLARATION OF ORIGINALITY

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DECLARATION OF ORIGINALITY

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CURRICULUM VITAE

TABLE OF CONTENTS

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I. INTRODUCTION

II. LITERATURE REVIEW

III. MATERIALS AND METHODS

IV. RESULTS AND DISCUSSION

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