Institutional Repository | Satya Wacana Christian University: Tocotrienol Rich Fraction (TRF) and Curcumin Extract in Fixing the Fat and Cholesterol Profile of Mice (BALB/C) Blood Serum
Tocotrienol Rich Fraction (TRF) and Curcumin
Extract in Fixing the Fat and Cholesterol Profile of
Mice (BALB/C) Blood Serum
Tesis
Diajukan kepada
Program Studi Magister Biologi
Untuk memperoleh gelar Magister Sains (M.Si)
Oleh:
Widiyasri Lodu
NIM : 422014002
Program Studi Magister Biologi
Universitas Kristen Satya Wacana
Salatiga
2017
Kata Pengantar
Puji syukur kepada Tuhan Yang Maha Esa karena atas
hikmat, berkat dan rahmatNya penulis dapat menyelesaikan
studi dan tesis yang berjudul “Tocotrienol Rich Fraction
(TRF) and Curcumin Extract In Fixing The Fat and
Cholesterol Profile Of Mice (Balb/C) Blood Serum”.
Penulis mengucapkan terima kasih kepada :
1. Rektor Universitas Kristen Satya Wacana, Prof. Pdt. John
A. Titaley, Th.D., atas kesempatan berharga yang
diberikan untuk studi lanjut di Magister Biologi
Universitas Kristen Satya Wacana.
2. Ir. Ferry F. Karwur, M.Sc., Ph.D., selaku Ketua Program
Studi Magister Biologi sekaligus pembimbing Tesis yang
telah membimbing secara intensif serta memberikan
perhatian dan motivasi selama penulis studi di Magister
Biologi.
3. Segenap jajaran dosen Magister Biologi UKSW yang telah
mencurahkan ilmu selama penulis studi di MB UKSW.
4. Keluarga, Papa dan Mama serta kakak (k’ Fred, k’ Son, k’
Elvis),Icha dan Agung yang selalu memberikan kasih
sayang, dukungan moril dan materi, nasihat, dan semua
doa yang selalu memberikan kekuatan bagi penulis.
5. Ibu Anastasia Natalia Kurniasari, S.Si., selaku sekretariat
di Program Studi Magister Biologi UKSW yang banyak
membantu dalam hal administrasi.
6. Laboran Lab. CARC (Carotenoid and Antioxidant Research
Center), Kak Norson Totoda “Ocon”, atas pendampingan
dan motivasi selama di Laboratorium.
7. Sahabat seangkatan MB 2014 (Mat, Dwi, Bu Sisil, Bu
Hesty, K’ Mety) yang memberikan makna persahabatan
dan saling mendukung.
Lalu teman lainnya di MB UKSW, k’ Dika, Fitri, Kristin,
James, Reni, Prima, k’opo, k’Yos, Eni, Ani, Abe, Petra
terimakasih atas setiap kebersamaan untuk saling
menguatkan dan diskusi ilmiahnya.
8. Linnaaw Student center rumah kedua tempat saya
berkomunitas, bertumbuh dalam Iman dan tempat
mengasah kemampuan berbahasa inggris. Terimakasih
dr. Jay Angeles dan Bu Jen yang sudah menerima saya
bergabung dalam setiap pelayanan, sudah menjadi
panutan dan orang tua saya selama di Salatiga serta bro
Alemar Amdengan dan k’ Liezle Fuertes atas semua
nasehat, motivasi dan bantuannya.
9. Panti asuhan terang anak bangsa, Mami Priska dan adikadik
panti
untuk
kebersamaan,
motivasi
dan
penerimaannya selama saya melayani disana.
10. Teman-teman diskusi LSC yang selalu memberikan
motivasi, value dan keceriaan dalam setiap situasi,
Joshua, Yuls, Matheus A, Dedek, Taha, En, Bags, Adi, Ge,
Tasya, En.
Penulis menyadari bahwa dalam penulisan tesis ini
masih jauh dari sempurna, sehingga penulis sangat
mengharapkan
saran
dan
kritik
konstruktif
demi
penyempurnaan karya ini serta agar dapat berguna bagi
perkembangan ilmu pengetahuan dan penulisan karya-karya
ilmiah selanjutnya. Semoga karya ini dapat bermanfaat bagi
kemajuan penelitian Indonesia. Segala hormat dan kemuliaan
hanya bagi Tuhan.
Salatiga, 21 November 2017
Widiyasri Lodu
Abstrak
Peningkatan kadar kolesterol adalah salah satu pemicu
penyakit kardiovaskuler oleh sebab itu penelitian - penelitian
yang berhubungan dengan penurunan kadar kolesterol darah
terus berkembangan tak terkecuali penelitian bahan alam
yang berdaya guna farmasi seperti kurkumin dan Tokotrienol
Rich Fraction yang telah banyak dilaporkan dalam
kebermanfaatannya sebagai antioksidan, dan mampu
menurunkan kolesterol. Tujuan dari penelitian ini adalah
untuk mengetahui pengaruh TRF dan ekstrak curcumin
terhadap kadar trigliserida, HDL,LDL dan, kolesterol total.
Penelitian ini merupakan penelitian eksperimen dengan
rancangan acak kelompok. Dalam penelitian ini digunakan 25
ekor mencit galur Balb/C dibagi menjadi 5 kelompok secara
acak yang diberi perlakuan selama 5 minggu. (K-) diberi diet
standar, (K+) diberi diet standar dan tinggi kolesterol. (P1)
diberi diet standar, tinggi kolesterol dan TRF, (P2) diberi diet
standar, tinggi kolesterol dan curcumin, dan (P3) diberi diet
standar, tinggi kolesterol , kombinasi TRF, dan kurkumin.
Kadar kolesterol diukur menggunakan metode CHODPAP,
data dianalisis menggunakan One Way ANOVA dan Tukey
HSD. Hasil perhitungan kadar trigliserida, kadar kolesterol
HDL, LDL dan, kolesterol total menunjukkan nilai (p=0,000).
Perlakuan menunjukkan beda nyata terhadap kontrol. Dosis
P2 terbukti menurunkan kadar kolesterol LDL hingga 61.8
mg/dL dibandingkan (K+)81.03mg/dL dan (K-) 128.69 mg/dL,
dan menurunkan kolesterol total hingga 134.33 mg/dL
dibandingkan K(+)196.33 mg/dL dan (K-) 156.66 mg/dL ,
kadar kolesterol HDL efektif meningkat dalam perlakuan P3
(65.14 mg/dL) dibandingkan (K+)41.77 mg/dL dan (K-) 31.66
mg/dL dan, P1 terbukti menurunkan kadar trigliserida hingga
120 mg/dL dibandingkN (K+)180.66 mg/dL dan (K-) 169.33
mg/dL.
Kata kunci: TRF, curcumin, trigliserida, HDL,LDL, kolesterol
total
Abstract
The increase of cholesterol levels is one trigger of
cardiovascular diseases. Therefore, studies related to the
decrease in blood cholesterol levels continue to develop as well
as studies of pharmaceutical natural ingredients such as
curcumin and Tocotrienol Rich Fraction (TFR) which have been
widely reported in its usefulness as an antioxidant, and its
ability to lower cholesterol. The purpose of this study is to
determine the effect of TRF and curcumin extract on the levels
of triglyceride, HDL, LDL and total cholesterol. This is an
experimental research using a randomized posttest only
control group design. In this research, 25 Balb/C mice were
randomly divided into 5 groups and treated for 5 weeks. (K-)
were given a standard diet, (K+) were given a standard diet and
high cholesterol. (P1) were given a standard diet, high
cholesterol and CURCUMIN, and (P3) were given a standard
diet, high cholesterol, a combination of TRF and curcumin.
Cholesterol levels were measured using the CHODPAP method
and the data were analyzed using One Way ANOVA and Tukey
HSD. The calculated results of triglyceride levels, HDL
cholesterol, LDL and cholesterol levels showed a value of (p =
0.000). The treatment showed a significant difference of the
control. The P2 dose proved to lower the LDL cholesterol levels
up to 61.8 mg/dL compared to (K+) which was 81.03mg/dL
and (K-) 128.69 mg/dL, and lowered the total cholesterol by
134.33 mg/dL compared to K(+) which was 196.33 mg/dL and
(K-) 156.66 mg/dL, effective HDL cholesterol levels increased
in P3 treatment (65.14 mg/dL) compared to (K+) 41.77 mg/dL
and (K-) 31.66 mg/dL and, P1 was shown to decrease
triglyceride levels to 120 mg/dL compared to (K+) 180.66
mg/dL and (K-) 169.33 mg/dL.
Key words: TRF, Curcumin, triglyceride, HDL, LDL, total
cholesterol
Daftar Isi
Kata Pengantar .................................................................... v
Daftar Isi ........................................................................... vii
Abstrak .............................................................................. viii
Abstract ............................................................................... ix
Riset Paper
Tocotrienol Rich Fraction (TRF) and Curcumin Extract
In Fixing The Fat and Cholesterol Profile Of Mice
(Balb/C) Blood Serum .......................................................... 1
Review Paper 1
Mode of action Of tocotrienol as anticancer .......................... 2
Mini Riset
Pemisahan dan Identifikasi Isomer Tokotrienol
Dari Tokotrienol Rich Fraction (TRF) .................................... 3
Ilmiah Populer
Peluang Industri Farmasi Berbahan Baku Sawit:
Tokotrienol, Kolesterol dan Kanker ...................................... 4
Tocotrienol Rich Fraction (TRF) and Curcumin Extract in
Fixing the Fat and Cholesterol Profile of Mice (BALB/C)
Blood Serum
Widiyasri Lodu1*, Ferry Fredy Karwur1,2*
1Graduate
Student Master of Biology Program, Universitas
Satya Wacana, Salatiga, Jawa Tengah
Kristen
2Lecturer
Faculty of Medicine and Health Science, Universitas
Kristen Satya Wacana, Salatiga, Jawa Tengah
Email : [email protected]
Abstract
The increase of cholesterol levels is one trigger of cardiovascular
diseases. Therefore, studies related to the decrease in blood
cholesterol levels continue to develop as well as studies of
pharmaceutical natural ingredients such as Curcumin and
Tocotrienol Rich Fraction (TFR) which have been widely reported in
its usefulness as an antioxidant, and its ability to lower cholesterol.
The purpose of this study is to determine the effect of TRF and
CURCUMIN extract on the levels of triglyceride, HDL, LDL and total
cholesterol. This is an experimental research using a randomized
posttest only control group design. In this research, 25 Balb/C mice
were randomly divided into 5 groups and treated for 5 weeks. (K-)
were given a standard diet, (K+) were given a standard diet and high
cholesterol. (P1) were given a standard diet, high cholesterol and
CURCUMIN, and (P3) were given a standard diet, high cholesterol, a
combination of TRF and curcumin. Cholesterol levels were measured
using the CHODPAP method and the data were analyzed using One
Way ANOVA and Tukey HSD. The calculated results of triglyceride
levels, HDL cholesterol, LDL and cholesterol levels showed a value of
(p = 0.000). The treatment showed a significant difference of the
control. The P2 dose proved to lower the LDL cholesterol levels up to
61.8 mg/dL compared to (K+) which was 81.03mg/dL and (K-)
128.69 mg/dL, and lowered the total cholesterol by 134.33 mg/dL
compared to K(+) which was 196.33 mg/dL and (K-) 156.66 mg/dL,
effective HDL cholesterol levels increased in P3 treatment (65.14
mg/dL) compared to (K+) 41.77 mg/dL and (K-) 31.66 mg/dL and,
P1 was shown to decrease triglyceride levels to 120 mg/dL compared
to (K+) 180.66 mg/dL and (K-) 169.33 mg/dL.
Key words: TRF, CURCUMIN, Triglyceride, HDL, LDL, Total cholesterol
Introduction
Cardiovascular disease is the first killer disease in the
world. According to the WHO data in 2015, an estimation of 17.7%
people died from cardiovascular, representing 31% of the world's
population deaths. Meanwhile the prevalence of cardiovascular
disease (coronary heart disease, heart failure and stroke) in
Indonesia, based on the data of Health Research base 2013, is 0.5%
or approximately 883,447 people, whereas based on the symptom’s
diagnosis there are 1.5% or an estimation of 2,650,340 people.
There are two risk factors causing the occurrence of cardiovascular
disease, which are non-modified risk factor and modified risk factor.
Non modified risk factors include age, gender, race, and family
history, while the modified risk factors are elevated levels of serum
lipids, hypertension, smoking, impaired glucose tolerance, and a
high saturated fat, cholesterol and calories diet (Price et al., 1995).
Of these two risk factors, hypertension and a high-fat diet are the
two most important risk factors of cardiovascular disease (Ying et
al., 2011; Fuchs et al., 2012). A high-fat diet as one of the risk
factors of cardiovascular disease will affect the body’s lipid profile.
The lipid profile in the blood consists of various fractions including
the total cholesterol, HDL cholesterol, triglycerides and LDL
cholesterol (Tacikowski et al., 2005). Cholesterol is indeed needed by
the body but excessive cholesterol will be harmful for the body thus
many studies have been conducted which dealt with natural
ingredients that are safe and effective to prevent excess lipid profile
in the body. One of the natural ingredients reported is curcumin
which is used as antioxidant, anti-inflammatory and antihypercholesterolemia (Peschel et al., 2006). Curcumin is effective in
controlling cholesterol and potentially reduces cardiovascular
complications resulting from hypercholesterolemia and could protect
patients at risk of cardiovascular disease (Hussein et al. 2014).
Another study reported by Kapoor et al (2008) explains that the
treatment by using curcumin on white mice showed a significant
decrease of total cholesterol, triglyceride and LDL-C levels, while also
showing a significant increase of HDL-C levels. In addition to
curcumin, other natural ingredients such as Tocotrienol Rich
Fraction (TRF) have also been reported to be beneficial in inhibiting
cholesterol levels through an enzyme called HMG-Coa via post
transcription (Parket et al., 1993), the inhibition of cholesterol levels
through HMG-Coa enzymes via post transcription can control the
risk of coronary heart disease in the human hypercholesterolemia
subjects (Qureshi et al., 2001). A TRF single treatment can reduce
the risk of atherosclerosis (Nafeesa et al., 2001). Besides the single
treatment, a combination of TRF and lovastatin is reported to lower
cholesterol (Qureshi et a., 2002).
Based on the description of health problems on the risk of
cardiovascular disease caused by high level of lipid products and
previous studies on the effectiveness of single treatment of TRF and
curcumin in inhibiting and reducing excessive lipid products in the
body, this study aims to identify the effect of giving TRF and
curcumin extract by either single or combination treatments in
improving the lipid profile of BALB/C mice.
MATERIALS AND METHODS
A. Materials
Experimental Materials
The tocotrienol rich fraction (TRF) was obtained from the Davos
Life Pte. Ltd. Singapore, and the standard food HI-PRO-VITE
592, high cholesterol diet, and CURCUMIN were obtained from
the result of turmeric rhizome extract conducted at the
Laboratory of Carotenoid and Antioxidant Research Center
Universitas Kristen Satya Wacana Salatiga.
Reagen Kit Cholesterol from Boehringer Mannheimm GmbH
Diagnostica; Reagen Kit HDL Total from Merck; Reagen Kit
triglyceride Total from Merck; ; EDTA tube; alcohol 70%;
ethanol ; hexane.
Experimental Animal
4-week Balb/C male mice obtained from the
Medicine, Universitas Diponegoro, Semarang.
acclimatized for 2 weeks in order to familiarize
environment and the new treatment. At this stage,
of the general state and weight were done.
Faculty of
Mice were
it with the
observation
Test Materials Preparation
The TRF obtained were TRF that have been purified therefore
the TRF can be directly used without going through the
extraction process. The extracted test material was curcumin.
A total of 20 grams of turmeric were set into a three-necked
flask, then extracted with 50%, 70% and 96% of ethanol with
the ratio of raw materials: solvent (1:4) b/v, for 60, 120 and
180 minutes with extraction stages of two and three stages.
The obtained rhinatate was then distilled and analyzed using
TLC and UV-spectrophotometer - a duplicate of Variant Cary
50 was seen. In addition to TRF and CURCUMIN ingredients,
high-fat diet foods were used, the ingredients used were lard
and yolk. The technique of making high-fat feed suspension
was by mixing 50gr standard food, 100gr egg yolks, and 50gr of
pork fat. The formed dough was molded into a pellet then put
in the oven.
B. Method
The research was conducted at the Laboratory of Carotenoid
and Antioxidant Research Center, Universitas Kristen Satya
Wacana, Salatiga. Time of research was February – August
2017. The research variables were free variables of TRF and
CURCUMIN; the bound variables were total cholesterol, HDL
cholesterol, LDL cholesterol, and blood plasma triglyceride of
mice; Control variables were strain and sex.
Experimental design and treatment
This research used a randomized posttest only control group
design. The trial animals were 4 weeks old Balb/c mice,
weighing 20-50 gr with a total of 25 (twenty five) mice divided
randomly into 5 treatments, each of which consisted of five
mice with 5 replicates each labeled with different colors on the
tail. The first treatment was the normal control by giving the
standard diet, the second treatment was the control treatment
of giving the high cholesterol and standard diet, the treatment
of III, IV and V respectively acquired the same high cholesterol
diet with the control group and were treated with oral test
materials in a dose of TRF 0,208 mg/20g Weight/day,
Curcumin 0,3 mg/20 g Weight/day and a combination dose
of TRF + curcumin 0,5 mg/20 g Weight/day. The standard
diet is given twice daily with ad libitum drinking water, the
test material was given orally (with gastric tube) 1 mL every
afternoon for 4 weeks. After four weeks of treatment, the mice
were fasted for 12 hours where after the fasting period 3 mice
were selected each with different labels, and then the blood of
the mice were taken through the vein of the tail, and the
triglyceride, HDL, LDL and total cholesterol were measured by
using the CHOD-PAP method. The data of the research were
analyzed by variance analysis (ANOVA), and if the analysis of
variance result showed real effect, it will be continued with
the TUKEY HSD test of 95% significance level.
Measurement of total cholesterol levels
The mice blood serum total cholesterol examination was
measured using CHOD-PAP (Cholesterol Oxidase Para
Aminophenazone). 10 μL sample was mixed with 1000 μL
cholesterol reagent and left at room temperature for 20
minutes. The absorbance was measured on a 500nm λ
spectrophotometer with blank solution as point 0, the blank
solution of 10 μL aqua bidest was mixed with 1000 μL
standard reagent. Calculation of the total cholesterol
concentration is by the following formula:
Measurement of HDL levels
A serum of 20 μL with 1000 μL precipitation reagent was
inserted into a centrifuge at the speed of 2500g for 20
minutes. Supernatant was used for the measurement of HDL
cholesterol levels. Supernatant plus 1000 μL reagent
precipitation of HDL, was left at room temperature for 10
minutes, after that it was read at λ 500nm with a blank
solution as point 0, the blank solution of 10 μL aqua bidest
was mixed with 1000 μL standard reagent. The calculation of
HDL cholesterol concentration is by the following formula:
Measurement of triglyceride levels
A serum of 10 μL was mixed with 1000 μL of triglyceride
precipitation reagent, and was left at room temperature for 10
minutes. The absorbance was measured on a 500nm λ
spectrophotometer with a blank solution as point 0, then a
blank solution of 10 μL aqua bidest was mixed with 1000 μL
standard reagent. Calculation of the total cholesterol
concentration is with the following formula:
)
Measurement of LDL levels
The concentration of LDL cholesterol (LDL-C) was calculated
from the total levels of cholesterol (TC), HDL-cholesterol (HDL), and triglyceride (TG) according to the Fried & Wald formula:
LDL-C = TC – (HDL-C) – TG/5 mg/dl.
RESULTS AND DISCUSSION
The results of this research revealed that the effect combination
of Tocotrienol Rich Fraction (TRF) and CURCUMIN extract
showed a significant decrease in triglyceride level, LDL
cholesterol level, total cholesterol level and an increase in HDL
cholesterol level of Balb/c white mice blood serum (p
Extract in Fixing the Fat and Cholesterol Profile of
Mice (BALB/C) Blood Serum
Tesis
Diajukan kepada
Program Studi Magister Biologi
Untuk memperoleh gelar Magister Sains (M.Si)
Oleh:
Widiyasri Lodu
NIM : 422014002
Program Studi Magister Biologi
Universitas Kristen Satya Wacana
Salatiga
2017
Kata Pengantar
Puji syukur kepada Tuhan Yang Maha Esa karena atas
hikmat, berkat dan rahmatNya penulis dapat menyelesaikan
studi dan tesis yang berjudul “Tocotrienol Rich Fraction
(TRF) and Curcumin Extract In Fixing The Fat and
Cholesterol Profile Of Mice (Balb/C) Blood Serum”.
Penulis mengucapkan terima kasih kepada :
1. Rektor Universitas Kristen Satya Wacana, Prof. Pdt. John
A. Titaley, Th.D., atas kesempatan berharga yang
diberikan untuk studi lanjut di Magister Biologi
Universitas Kristen Satya Wacana.
2. Ir. Ferry F. Karwur, M.Sc., Ph.D., selaku Ketua Program
Studi Magister Biologi sekaligus pembimbing Tesis yang
telah membimbing secara intensif serta memberikan
perhatian dan motivasi selama penulis studi di Magister
Biologi.
3. Segenap jajaran dosen Magister Biologi UKSW yang telah
mencurahkan ilmu selama penulis studi di MB UKSW.
4. Keluarga, Papa dan Mama serta kakak (k’ Fred, k’ Son, k’
Elvis),Icha dan Agung yang selalu memberikan kasih
sayang, dukungan moril dan materi, nasihat, dan semua
doa yang selalu memberikan kekuatan bagi penulis.
5. Ibu Anastasia Natalia Kurniasari, S.Si., selaku sekretariat
di Program Studi Magister Biologi UKSW yang banyak
membantu dalam hal administrasi.
6. Laboran Lab. CARC (Carotenoid and Antioxidant Research
Center), Kak Norson Totoda “Ocon”, atas pendampingan
dan motivasi selama di Laboratorium.
7. Sahabat seangkatan MB 2014 (Mat, Dwi, Bu Sisil, Bu
Hesty, K’ Mety) yang memberikan makna persahabatan
dan saling mendukung.
Lalu teman lainnya di MB UKSW, k’ Dika, Fitri, Kristin,
James, Reni, Prima, k’opo, k’Yos, Eni, Ani, Abe, Petra
terimakasih atas setiap kebersamaan untuk saling
menguatkan dan diskusi ilmiahnya.
8. Linnaaw Student center rumah kedua tempat saya
berkomunitas, bertumbuh dalam Iman dan tempat
mengasah kemampuan berbahasa inggris. Terimakasih
dr. Jay Angeles dan Bu Jen yang sudah menerima saya
bergabung dalam setiap pelayanan, sudah menjadi
panutan dan orang tua saya selama di Salatiga serta bro
Alemar Amdengan dan k’ Liezle Fuertes atas semua
nasehat, motivasi dan bantuannya.
9. Panti asuhan terang anak bangsa, Mami Priska dan adikadik
panti
untuk
kebersamaan,
motivasi
dan
penerimaannya selama saya melayani disana.
10. Teman-teman diskusi LSC yang selalu memberikan
motivasi, value dan keceriaan dalam setiap situasi,
Joshua, Yuls, Matheus A, Dedek, Taha, En, Bags, Adi, Ge,
Tasya, En.
Penulis menyadari bahwa dalam penulisan tesis ini
masih jauh dari sempurna, sehingga penulis sangat
mengharapkan
saran
dan
kritik
konstruktif
demi
penyempurnaan karya ini serta agar dapat berguna bagi
perkembangan ilmu pengetahuan dan penulisan karya-karya
ilmiah selanjutnya. Semoga karya ini dapat bermanfaat bagi
kemajuan penelitian Indonesia. Segala hormat dan kemuliaan
hanya bagi Tuhan.
Salatiga, 21 November 2017
Widiyasri Lodu
Abstrak
Peningkatan kadar kolesterol adalah salah satu pemicu
penyakit kardiovaskuler oleh sebab itu penelitian - penelitian
yang berhubungan dengan penurunan kadar kolesterol darah
terus berkembangan tak terkecuali penelitian bahan alam
yang berdaya guna farmasi seperti kurkumin dan Tokotrienol
Rich Fraction yang telah banyak dilaporkan dalam
kebermanfaatannya sebagai antioksidan, dan mampu
menurunkan kolesterol. Tujuan dari penelitian ini adalah
untuk mengetahui pengaruh TRF dan ekstrak curcumin
terhadap kadar trigliserida, HDL,LDL dan, kolesterol total.
Penelitian ini merupakan penelitian eksperimen dengan
rancangan acak kelompok. Dalam penelitian ini digunakan 25
ekor mencit galur Balb/C dibagi menjadi 5 kelompok secara
acak yang diberi perlakuan selama 5 minggu. (K-) diberi diet
standar, (K+) diberi diet standar dan tinggi kolesterol. (P1)
diberi diet standar, tinggi kolesterol dan TRF, (P2) diberi diet
standar, tinggi kolesterol dan curcumin, dan (P3) diberi diet
standar, tinggi kolesterol , kombinasi TRF, dan kurkumin.
Kadar kolesterol diukur menggunakan metode CHODPAP,
data dianalisis menggunakan One Way ANOVA dan Tukey
HSD. Hasil perhitungan kadar trigliserida, kadar kolesterol
HDL, LDL dan, kolesterol total menunjukkan nilai (p=0,000).
Perlakuan menunjukkan beda nyata terhadap kontrol. Dosis
P2 terbukti menurunkan kadar kolesterol LDL hingga 61.8
mg/dL dibandingkan (K+)81.03mg/dL dan (K-) 128.69 mg/dL,
dan menurunkan kolesterol total hingga 134.33 mg/dL
dibandingkan K(+)196.33 mg/dL dan (K-) 156.66 mg/dL ,
kadar kolesterol HDL efektif meningkat dalam perlakuan P3
(65.14 mg/dL) dibandingkan (K+)41.77 mg/dL dan (K-) 31.66
mg/dL dan, P1 terbukti menurunkan kadar trigliserida hingga
120 mg/dL dibandingkN (K+)180.66 mg/dL dan (K-) 169.33
mg/dL.
Kata kunci: TRF, curcumin, trigliserida, HDL,LDL, kolesterol
total
Abstract
The increase of cholesterol levels is one trigger of
cardiovascular diseases. Therefore, studies related to the
decrease in blood cholesterol levels continue to develop as well
as studies of pharmaceutical natural ingredients such as
curcumin and Tocotrienol Rich Fraction (TFR) which have been
widely reported in its usefulness as an antioxidant, and its
ability to lower cholesterol. The purpose of this study is to
determine the effect of TRF and curcumin extract on the levels
of triglyceride, HDL, LDL and total cholesterol. This is an
experimental research using a randomized posttest only
control group design. In this research, 25 Balb/C mice were
randomly divided into 5 groups and treated for 5 weeks. (K-)
were given a standard diet, (K+) were given a standard diet and
high cholesterol. (P1) were given a standard diet, high
cholesterol and CURCUMIN, and (P3) were given a standard
diet, high cholesterol, a combination of TRF and curcumin.
Cholesterol levels were measured using the CHODPAP method
and the data were analyzed using One Way ANOVA and Tukey
HSD. The calculated results of triglyceride levels, HDL
cholesterol, LDL and cholesterol levels showed a value of (p =
0.000). The treatment showed a significant difference of the
control. The P2 dose proved to lower the LDL cholesterol levels
up to 61.8 mg/dL compared to (K+) which was 81.03mg/dL
and (K-) 128.69 mg/dL, and lowered the total cholesterol by
134.33 mg/dL compared to K(+) which was 196.33 mg/dL and
(K-) 156.66 mg/dL, effective HDL cholesterol levels increased
in P3 treatment (65.14 mg/dL) compared to (K+) 41.77 mg/dL
and (K-) 31.66 mg/dL and, P1 was shown to decrease
triglyceride levels to 120 mg/dL compared to (K+) 180.66
mg/dL and (K-) 169.33 mg/dL.
Key words: TRF, Curcumin, triglyceride, HDL, LDL, total
cholesterol
Daftar Isi
Kata Pengantar .................................................................... v
Daftar Isi ........................................................................... vii
Abstrak .............................................................................. viii
Abstract ............................................................................... ix
Riset Paper
Tocotrienol Rich Fraction (TRF) and Curcumin Extract
In Fixing The Fat and Cholesterol Profile Of Mice
(Balb/C) Blood Serum .......................................................... 1
Review Paper 1
Mode of action Of tocotrienol as anticancer .......................... 2
Mini Riset
Pemisahan dan Identifikasi Isomer Tokotrienol
Dari Tokotrienol Rich Fraction (TRF) .................................... 3
Ilmiah Populer
Peluang Industri Farmasi Berbahan Baku Sawit:
Tokotrienol, Kolesterol dan Kanker ...................................... 4
Tocotrienol Rich Fraction (TRF) and Curcumin Extract in
Fixing the Fat and Cholesterol Profile of Mice (BALB/C)
Blood Serum
Widiyasri Lodu1*, Ferry Fredy Karwur1,2*
1Graduate
Student Master of Biology Program, Universitas
Satya Wacana, Salatiga, Jawa Tengah
Kristen
2Lecturer
Faculty of Medicine and Health Science, Universitas
Kristen Satya Wacana, Salatiga, Jawa Tengah
Email : [email protected]
Abstract
The increase of cholesterol levels is one trigger of cardiovascular
diseases. Therefore, studies related to the decrease in blood
cholesterol levels continue to develop as well as studies of
pharmaceutical natural ingredients such as Curcumin and
Tocotrienol Rich Fraction (TFR) which have been widely reported in
its usefulness as an antioxidant, and its ability to lower cholesterol.
The purpose of this study is to determine the effect of TRF and
CURCUMIN extract on the levels of triglyceride, HDL, LDL and total
cholesterol. This is an experimental research using a randomized
posttest only control group design. In this research, 25 Balb/C mice
were randomly divided into 5 groups and treated for 5 weeks. (K-)
were given a standard diet, (K+) were given a standard diet and high
cholesterol. (P1) were given a standard diet, high cholesterol and
CURCUMIN, and (P3) were given a standard diet, high cholesterol, a
combination of TRF and curcumin. Cholesterol levels were measured
using the CHODPAP method and the data were analyzed using One
Way ANOVA and Tukey HSD. The calculated results of triglyceride
levels, HDL cholesterol, LDL and cholesterol levels showed a value of
(p = 0.000). The treatment showed a significant difference of the
control. The P2 dose proved to lower the LDL cholesterol levels up to
61.8 mg/dL compared to (K+) which was 81.03mg/dL and (K-)
128.69 mg/dL, and lowered the total cholesterol by 134.33 mg/dL
compared to K(+) which was 196.33 mg/dL and (K-) 156.66 mg/dL,
effective HDL cholesterol levels increased in P3 treatment (65.14
mg/dL) compared to (K+) 41.77 mg/dL and (K-) 31.66 mg/dL and,
P1 was shown to decrease triglyceride levels to 120 mg/dL compared
to (K+) 180.66 mg/dL and (K-) 169.33 mg/dL.
Key words: TRF, CURCUMIN, Triglyceride, HDL, LDL, Total cholesterol
Introduction
Cardiovascular disease is the first killer disease in the
world. According to the WHO data in 2015, an estimation of 17.7%
people died from cardiovascular, representing 31% of the world's
population deaths. Meanwhile the prevalence of cardiovascular
disease (coronary heart disease, heart failure and stroke) in
Indonesia, based on the data of Health Research base 2013, is 0.5%
or approximately 883,447 people, whereas based on the symptom’s
diagnosis there are 1.5% or an estimation of 2,650,340 people.
There are two risk factors causing the occurrence of cardiovascular
disease, which are non-modified risk factor and modified risk factor.
Non modified risk factors include age, gender, race, and family
history, while the modified risk factors are elevated levels of serum
lipids, hypertension, smoking, impaired glucose tolerance, and a
high saturated fat, cholesterol and calories diet (Price et al., 1995).
Of these two risk factors, hypertension and a high-fat diet are the
two most important risk factors of cardiovascular disease (Ying et
al., 2011; Fuchs et al., 2012). A high-fat diet as one of the risk
factors of cardiovascular disease will affect the body’s lipid profile.
The lipid profile in the blood consists of various fractions including
the total cholesterol, HDL cholesterol, triglycerides and LDL
cholesterol (Tacikowski et al., 2005). Cholesterol is indeed needed by
the body but excessive cholesterol will be harmful for the body thus
many studies have been conducted which dealt with natural
ingredients that are safe and effective to prevent excess lipid profile
in the body. One of the natural ingredients reported is curcumin
which is used as antioxidant, anti-inflammatory and antihypercholesterolemia (Peschel et al., 2006). Curcumin is effective in
controlling cholesterol and potentially reduces cardiovascular
complications resulting from hypercholesterolemia and could protect
patients at risk of cardiovascular disease (Hussein et al. 2014).
Another study reported by Kapoor et al (2008) explains that the
treatment by using curcumin on white mice showed a significant
decrease of total cholesterol, triglyceride and LDL-C levels, while also
showing a significant increase of HDL-C levels. In addition to
curcumin, other natural ingredients such as Tocotrienol Rich
Fraction (TRF) have also been reported to be beneficial in inhibiting
cholesterol levels through an enzyme called HMG-Coa via post
transcription (Parket et al., 1993), the inhibition of cholesterol levels
through HMG-Coa enzymes via post transcription can control the
risk of coronary heart disease in the human hypercholesterolemia
subjects (Qureshi et al., 2001). A TRF single treatment can reduce
the risk of atherosclerosis (Nafeesa et al., 2001). Besides the single
treatment, a combination of TRF and lovastatin is reported to lower
cholesterol (Qureshi et a., 2002).
Based on the description of health problems on the risk of
cardiovascular disease caused by high level of lipid products and
previous studies on the effectiveness of single treatment of TRF and
curcumin in inhibiting and reducing excessive lipid products in the
body, this study aims to identify the effect of giving TRF and
curcumin extract by either single or combination treatments in
improving the lipid profile of BALB/C mice.
MATERIALS AND METHODS
A. Materials
Experimental Materials
The tocotrienol rich fraction (TRF) was obtained from the Davos
Life Pte. Ltd. Singapore, and the standard food HI-PRO-VITE
592, high cholesterol diet, and CURCUMIN were obtained from
the result of turmeric rhizome extract conducted at the
Laboratory of Carotenoid and Antioxidant Research Center
Universitas Kristen Satya Wacana Salatiga.
Reagen Kit Cholesterol from Boehringer Mannheimm GmbH
Diagnostica; Reagen Kit HDL Total from Merck; Reagen Kit
triglyceride Total from Merck; ; EDTA tube; alcohol 70%;
ethanol ; hexane.
Experimental Animal
4-week Balb/C male mice obtained from the
Medicine, Universitas Diponegoro, Semarang.
acclimatized for 2 weeks in order to familiarize
environment and the new treatment. At this stage,
of the general state and weight were done.
Faculty of
Mice were
it with the
observation
Test Materials Preparation
The TRF obtained were TRF that have been purified therefore
the TRF can be directly used without going through the
extraction process. The extracted test material was curcumin.
A total of 20 grams of turmeric were set into a three-necked
flask, then extracted with 50%, 70% and 96% of ethanol with
the ratio of raw materials: solvent (1:4) b/v, for 60, 120 and
180 minutes with extraction stages of two and three stages.
The obtained rhinatate was then distilled and analyzed using
TLC and UV-spectrophotometer - a duplicate of Variant Cary
50 was seen. In addition to TRF and CURCUMIN ingredients,
high-fat diet foods were used, the ingredients used were lard
and yolk. The technique of making high-fat feed suspension
was by mixing 50gr standard food, 100gr egg yolks, and 50gr of
pork fat. The formed dough was molded into a pellet then put
in the oven.
B. Method
The research was conducted at the Laboratory of Carotenoid
and Antioxidant Research Center, Universitas Kristen Satya
Wacana, Salatiga. Time of research was February – August
2017. The research variables were free variables of TRF and
CURCUMIN; the bound variables were total cholesterol, HDL
cholesterol, LDL cholesterol, and blood plasma triglyceride of
mice; Control variables were strain and sex.
Experimental design and treatment
This research used a randomized posttest only control group
design. The trial animals were 4 weeks old Balb/c mice,
weighing 20-50 gr with a total of 25 (twenty five) mice divided
randomly into 5 treatments, each of which consisted of five
mice with 5 replicates each labeled with different colors on the
tail. The first treatment was the normal control by giving the
standard diet, the second treatment was the control treatment
of giving the high cholesterol and standard diet, the treatment
of III, IV and V respectively acquired the same high cholesterol
diet with the control group and were treated with oral test
materials in a dose of TRF 0,208 mg/20g Weight/day,
Curcumin 0,3 mg/20 g Weight/day and a combination dose
of TRF + curcumin 0,5 mg/20 g Weight/day. The standard
diet is given twice daily with ad libitum drinking water, the
test material was given orally (with gastric tube) 1 mL every
afternoon for 4 weeks. After four weeks of treatment, the mice
were fasted for 12 hours where after the fasting period 3 mice
were selected each with different labels, and then the blood of
the mice were taken through the vein of the tail, and the
triglyceride, HDL, LDL and total cholesterol were measured by
using the CHOD-PAP method. The data of the research were
analyzed by variance analysis (ANOVA), and if the analysis of
variance result showed real effect, it will be continued with
the TUKEY HSD test of 95% significance level.
Measurement of total cholesterol levels
The mice blood serum total cholesterol examination was
measured using CHOD-PAP (Cholesterol Oxidase Para
Aminophenazone). 10 μL sample was mixed with 1000 μL
cholesterol reagent and left at room temperature for 20
minutes. The absorbance was measured on a 500nm λ
spectrophotometer with blank solution as point 0, the blank
solution of 10 μL aqua bidest was mixed with 1000 μL
standard reagent. Calculation of the total cholesterol
concentration is by the following formula:
Measurement of HDL levels
A serum of 20 μL with 1000 μL precipitation reagent was
inserted into a centrifuge at the speed of 2500g for 20
minutes. Supernatant was used for the measurement of HDL
cholesterol levels. Supernatant plus 1000 μL reagent
precipitation of HDL, was left at room temperature for 10
minutes, after that it was read at λ 500nm with a blank
solution as point 0, the blank solution of 10 μL aqua bidest
was mixed with 1000 μL standard reagent. The calculation of
HDL cholesterol concentration is by the following formula:
Measurement of triglyceride levels
A serum of 10 μL was mixed with 1000 μL of triglyceride
precipitation reagent, and was left at room temperature for 10
minutes. The absorbance was measured on a 500nm λ
spectrophotometer with a blank solution as point 0, then a
blank solution of 10 μL aqua bidest was mixed with 1000 μL
standard reagent. Calculation of the total cholesterol
concentration is with the following formula:
)
Measurement of LDL levels
The concentration of LDL cholesterol (LDL-C) was calculated
from the total levels of cholesterol (TC), HDL-cholesterol (HDL), and triglyceride (TG) according to the Fried & Wald formula:
LDL-C = TC – (HDL-C) – TG/5 mg/dl.
RESULTS AND DISCUSSION
The results of this research revealed that the effect combination
of Tocotrienol Rich Fraction (TRF) and CURCUMIN extract
showed a significant decrease in triglyceride level, LDL
cholesterol level, total cholesterol level and an increase in HDL
cholesterol level of Balb/c white mice blood serum (p