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
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.
Alemar Amdengan dan k’ Liezle Fuertes atas semua nasehat, motivasi dan bantuannya.
9. Panti asuhan terang anak bangsa, Mami Priska dan adik-adik 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
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.
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.
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
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 Kristen
Satya Wacana, Salatiga, Jawa Tengah
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.
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, inflammatory and anti-hypercholesterolemia (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).
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 Faculty of Medicine, Universitas Diponegoro, Semarang. Mice were acclimatized for 2 weeks in order to familiarize it with the environment and the new treatment. At this stage, observation of the general state and weight were done.
Test Materials Preparation
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
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 <0.05) as shown in table 1. The results are described as follows:
Triglycerides Cholesterol Levels
The results showed that the triglyceride level in the TRF single treatment (P1) decreased significantly by 120.00 ± 8.88mg/dL, the combination (P3) was 124.33 ± 14.43 mg/dL and the treatment of CURCUMIN (P2) was 133.00 ± 6.24 mg/dL. The treatment group showed an evident difference of the high-fat diet (K +) group of 180.66 ± 1.15 mg/dL, and the control without high-fat treatment with concentration of 163.33 ± 3.05mg / dL. According to Mayes (2003), triglyceride is the main lipid storage in adipose tissue, this form of lipid will be released after the hydrolysis of the lipase enzyme turns into glycerol-free fatty acids. Free fatty acids will be bound to the serum albumin and used to transport to the tissues, where the fatty acids are used as an important fuel source. The high triglyceride level is associated with the increased risk of ischemic stroke from three to four times (Freiberg et al., 2008). In this study, a single treatment of TRF and CURCUMIN or a combination of both significantly decreased the levels of triglyceride cholesterol.
HDL Cholesterol Levels
curcumin (P3) significantly increased the HDL cholesterol level to 65.14 mg/dL, TRF single treatment (P1) of 56.13 mg/ dL and, curcumin single treatment (P2) of 45.93 mg / dL. High density lipoprotein (HDL) is a lipoprotein that removes lipids from the periphery to the liver. Because the molecule is relatively small compared to other lipoproteins, HDL can pass through the vascular endothelial cells and into the intima to transport back the cholesterol collected in the macrophages. Low levels of HDL in the blood will increase the risk of atherosclerosis and coronary heart disease (Moeliandari et al., 2002). In the results of this study a combination of TRF and CURCUMIN showed a very significant number in increasing HDL cholesterol levels followed by a single treatment of TRF and Curcumin.
LDL Cholesterol Levels
The single Curcumin (P2) treatment was able to significantly reduce the LDL level to 61.80 mg/dL, the combination (P3) was able to significantly decrease it to 64.66 mg/dL and the TRF (P1) treatment to 73.88 mg/dL. The three treatment groups showed a significant difference to the control group without high lipid treatment (K-) of 81.03 mg/dL and high lipid food control (K +) of 128.40 mg/dL. Low Density Lipoproteins are lipoproteins that transport lipids from the liver to the periphery. According to Murray et al (1996), LDL contains half to two thirds of cholesterol. High LDL levels are at risk of causing atherosclerosis (Klatt et al., 1996). In this research, single treatment of CURCUMIN significantly decreased the total cholesterol level which was followed by TRF combination treatment and curcumin and TRF single treatment.
Total Cholesterol Levels
The single treatment of KURKUMIN 0.3 mL/weight/day (P2) significantly decreased the total cholesterol level up to 134.33 mg/dL, which was significantly different from the control (+) of
the mice’s high lipid diet 193.33 mg/d, whereas the
from the body through the bile as cholesterol salt (Murray et al., 1999).
Table1. The effect of TRF, CURCUMIN and its combination on total cholesterol levels, HDL, triglycerides and LDL serum of mice blood in which were given high lipid foods.
DISCUSSION
which would increase the HDL cholesterol levels through
PPARα stimuli (Kang et al., 2009; Shiddiqui et al., 2006).
Whereas the antioxidant activity of TRF can increase the Lecithin Cholesterol Acyltransferase (LCAT) activity (Moradi et al., 2009), LCAT is an enzyme that can transform free cholesterol to cholesterol ester which forms new HDL. This can increase the HDL serum levels (Lewis, G.F, 2005). Tocotrienol also controls the production of cholesterol in mammalian cells through post transcription by suppressing HMG CoA reductase and specifically modulating intracellular mechanisms to control the degradation of protein reductase. This enzyme activity control has implications for the cholesterol synthesis and mevalonate line products control (Parker et al., 1996).
CONCLUSION
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