CYTOTOXICITY EFFECT OF CINNAMON OIL ON WiDr Vero CULTURE CELL AND SUB CRONIC TOXICITY EFFECT OF CINNAMON OIL ON ALTAST LEVELS MALE RAT Efek Sitotoksisitas Minyak Kayu Manis Terhadap Kultur Sel WiDr Vero Serta Efek Toksisitas Subkronis Minyak Kayu Manis Pa

  

CYTOTOXICITY EFFECT OF CINNAMON OIL ON WiDr & Vero CULTURE CELL AND

SUB CRONIC TOXICITY EFFECT OF CINNAMON OIL ON ALT/AST LEVELS MALE RAT

Efek Sitotoksisitas Minyak Kayu Manis Terhadap Kultur Sel

WiDr & Vero Serta Efek Toksisitas Subkronis Minyak Kayu Manis Pada Kadar ALT/

  

ASL Tikus Putih Jantan

W Herdwiani, F Leviana, Z Imama, Sari, AA Soemardji, Elfahmi, MI Tan

  

Faculty Of Pharmacy, Setia Budi University, Surakarta

School Of Pharmacy, Institut Of Technology Bandung

E-mail: herdwiani@gmail.com

  

ABSTRAK

Lebih dari 1 juta kasus kanker terjadi setiap tahun dan separuhnya berada di negara-negara berkembang. Pengem-

bangan pengobatan alternatif pada kanker saat ini menjadi perhatian penting dikarenakan kegagalan kemoterapi

konvensional. Karya ini telah membahas potensi antineoplastik Minyak Cinnamon kanker. Penelitian ini bertujuan

untuk mengetahui efek cytotoxity dari Cinnamon Oil On sel kolorektal budaya kanker (WiDr) dan normal Kultur Sel

(Vero) dan efek toksisitas subkronis minyak kayu manis Pada Pria Rat. Efek Sitotoxic dari WiDr sel kultur dan bu-

₅₀

daya Vero sel dilakukan oleh MTT assay, untuk mendapatkan nomor IC dan pengujian toksisitas subkronis meng-

gunakan 20 tikus jantan dan dibagi menjadi 4 kelompok. Setiap kelompok diberi perlakuan minyak kayu manis

dalam dosis perbedaan, pertama kelompok 50 mg / kg BB, kelompok kedua 100 mg / kg BB, kelompok ketiga 200

mg / kg BB dan kelompok terakhir kontrol negatif. Data diperoleh dari pemeriksaan SGPT / ALT nad SGOT / AST

sebelum dan sesudah perlakuan pada tanggal 1, 2, minggu ke-3 dan ke-4 dan tokoh histopatologi hepar pada akhir

minggu ke-4. Hasil Data dianalisis dengan menggunakan SPPS. Hasil penelitian menunjukkan bahwa minyak kayu

₅₀

manis memiliki IC WiDr dan Vero sel kultur 38,02 mg / ml dan 27,35 mg / ml masing-masing. Minyak Cinnamon

tidak memiliki efek toksisitas subkronis pada hepar tikus putih jantan.

  Kata kunci: kayu manis, sitotoksisitas, WiDr, toksisitas

ABSTRACT

  

More than 1 million cancer cases occur each year and half are in developing countries. Attention towards new

alternative was invited by the failure of conventional chemotheraphy. The present work has addressed the anti-

neoplastic potential of the Cinnamon Oil in cancer. This research aims to investigate cytotoxity effect of Cinnamon

Oil On colorectal cancer culture cells (WiDr) and normal Culture Cell (Vero) and subchronic toxicity effect of cin-

namon oil On Male Rat. Sitotoxic effect of WiDr culture cell and Vero culture cell were done by MTT assay, to get

an IC50 number and subchronic toxicity testing using 20 male rat and were divided into 4 groups. Each group was

given treatment of cinnamon oil in difference doses, first group 50 mg/kg BW, second group 100 mg/kg BW, third

group 200 mg/kg BW and the last group negative control. The data were obtained from the examination SGPT/

ALT nad SGOT/AST before and after treatment on 1st, 2nd, 3rd and 4th week and histopathological figures of the

hepar at the end of 4th week. The data results were analyzed using SPPS. The results showed that cinnamon oil

had an IC50 of WiDr and Vero culture cell 38,02 µg/ml and 27,35 µg/ml respectively. Cinnamon oil had no sub-

chronic toxicity effect on the hepar of white male rat.

  Keywords : cinnamon, citotoxicity, WiDr, Vero, toxicity

  INTRODUCTION

  MATERIALS AND METHOD Materials

  After 2 hr incubation at 37°C, the me- dium was removed and 200 ml of DMSO

  ., 2008). Briefly, the cells were plated at a density of 1x103 cells/well into 96 well plates. After 24 hr or 48 hr exposure to cinnamon oil (500 µg/ml, 250 µg/ml, 125 µg/ml, 62,5 µg/ml, 31,25 µg/ml, 15,65 µg/ml, 7,8 µg/ml, and 3,9 µg/ml) dissolved in DMSO, 20 uL MTT (5 mg/ ml) stock solution was added to each well.

  et al

  MTT assay was based on the mitocon- drial enzyme reduction of Tetrazolium dye to detect and determine cell viability (Zeytinoglu

  WiDr and Vero culture cell used were a collection of the Laboratory of Parasitology of the Faculty of Medicine, University of Gadjah Mada. Cells grown in culture medium grower Roswell Park Memorial Institute (RPMI) 1640 (Gibco) Fetal Bovine Serum containing (FBS) 10 % v/v (Gibco), penicillin - streptomycin 1 % v/v (Gibco) and Fungison 2% v/v (Gibco).

  Method Cytotoxic Activity

  The plant materials Cinamomum bark (Cinnamomum burmanni Nees ex Bl.) used in this study were purchased from Jambu Village, Ambarawa,Semarang, Central Java.

  ., 2009). Additionally the in vitro cytotoxic and apoptotic effect of the oil on normal Vero cells and WiDr cell lines were also been inves- tigated. This research aims to investigate sub- chronic toxicity effect of cinnamon oil on male rat and cytotoxicity effect of cinnamon oil on WiDr cell lines and Vero cell lines.

  Cancer is the second after heart disease as a leading cause of death in the United States and British (Dipiro, 2008). According to WHO, more than 1 million cases occur each year and more than half are in developing countries (Aapro, 2007). Cancer (a malignant tumor) is present when the tumor tissue destructively invades healthy surrounding tissue or when dislodged tumor cells form secondary tumors (metastases) in other organs (Heinz et al., 2000).

  al

  apoptosis/necrosis by interfering with normal mitochondrial function in yeast cells (Bakkali et al., 2006). Furthermore, cytotoxic and apoptotic activities of several constituents of cinnamon species have been shown in vari- ous cancer cells (Chen et al., 2007; Huang et al., 2007; Ka et al., 2003; Lee et al., 2004). Collec- tively, these data suggest that cinnamon could be proposed as a potent anticancer (Singh et

  camphora induced early apoptosis and late

  The essential oil from Cinnamomum

  Several studies have been conducted on herbs that possess anticancer properties and have been used as potent anticancer drugs. The present work has addressed the antineoplastic potential of the spice cinnamon in cancer. Cin- namon besides altering the growth kinetics of cells induces apoptosis through loss of mito- chondrial membrane potential.

  Attention towards new alternative was invited by the failure of conventional chemo- therapy to reduce mortality. Approaches that would reduce morbidity as well as side effects were conferred by conventional chemotherapy. A source of effective anticancer agents, plants have played a significant role. About 60% of currently used anticancer drugs are derived from natural sources such as plants, marine organisms and microorganisms (Rocha et al., 2001; Mann, 2002; Deorukhkar et al., 2007).

  Cytotoxic substances that particularly affect proliferating or dividing cells are cyto- statics. Not only retards tumor growth but also initiate apoptosis (programmed cell death) damage to mitotic processes. Because tissues have a physiologically high mitotic rate, so they can be affected by cytostatic therapy. There are adverse effects of cytostatic therapy i.e loss of hair, gastrointestinal disturbances, nause- vomiting, lowered resistance to infection and bone marrow depression (Heinz et al., 2000).

  W Herdwiani, F Leviana, Z Imama, Sari, AA Soemardji, Elfahmi, MI Tan

  CYTOTOXICITY EFFECT OF CINNAMON OIL ON WiDr & Vero CULTURE CELL AND SUB CRONIC

TOXICITY EFFECT OF CINNAMON OIL ON ALT/AST LEVELS MALE RAT

  Efek Sitotoksisitas Minyak Kayu Manis Terhadap Kultur Sel WiDr & Vero Serta Efek Toksisitas Subkronis Minyak Kayu Manis Pada Kadar ALT/ASL Tikus Putih Jantan

  were added to each well. Cells were incubated content more than 55% (Table 1). at 25°C for further 10 min and then the absor-

  Table 1. Calculation Levels of Cinnamaldehyde

  bance was read on a ELISA reader at a wave- _{Cinnamaldehyde Content Refference Conclusion} lenght of 540 nm. The values of the blank wells

  66,59 % >55 % qualify (Anonim, 2006b)

  were subracted from each well of treated and control cells, the percentage viability were de-

  Cytotoxicity effect of Cinnamon Oil on WiDr culture terminated as formulated below : cells and Vero cells.

  % viable cells= (the absorbance of treated cells - the absorbance of the blank) Figure 1 was viewed about the curve of (the absorbance of the control-the absorbance of the blank).

  Log Dose Vs % Viability cinnamon oils and

  Subcronic Toxicity Test

  Doxorubicin as positive control to WiDr cul- ture cells. White male rat (Rattus novergicus) was used in this research. This animal was pur- chased from Laboratory of Pharmacology Se- tia Budi University, Surakarta on March 2013.

  Subchronic toxicity testing using 20 male rats and were divided into 4 groups. Each group was treated with cinnamon oil in difference doses, first group 50 mg/kg BW, second group and

  third group 200 mg/kg BW

  100 mg/kg BW, the last group negative control. Data were ob-

  Figure 1. Curve comparison of the log-dose relation

  tained from the examination SGPT/ALT and

  ship versus percent living WiDr cells (cell vi

  SGOT/AST levels before and after treatment on _{st nd th}

  ability) with Doxorubicin treatment and cin 1 , 2 week and histopathological namon oil concentrations of 3,9-500 µg/ ml.

  , 3rd and 4 figures of the hepar and kidney at the end of _th Figure 2 Curve comparison of the log- week. The data results were analyzed using

  4 dose relationship versus percent living Vero SPPS software. cells (cell viability) with cinnamon oil concen- trations of 3,9-500 µg/ml. The IC50 value of

RESULT AND DISCUSSION

  cinnamon Oil on WiDr culture cell was 38,02 Isolation of the Cinnamon Oil. µg/ml. The plant was determinated by Mor- phology and Systematics Laboratory Setia Budi

  University. After it was taken its bark and was isolated their oil by destilation method. The rendement oil of cinnamon oil was 0,2 %. The identification of TLC of cinnamon oil was done with using silica gel stationary phase and mo- bile phase toluene: ethyl acetate (93:7) with an standard pure cinnamaldehyd. aldehyd 0,71. Identification of TLC showed positive results for the presence of cinnamaldehyd content.

  The active compounds cinnamaldehyde assay using Gas Chromatography. Provided that cinnamon oil contains active compounds Figure 2. Morphology WiDr cells (a. control cells, b.

  medium, c. Concentration of 3,9 µg/ml, d. concen-

  cinnamaldehyde was 66,45 % and the prevail-

  tration of 500 µg/ml)

  ing regulations that cinnamaldehyde

  Morphological observation of WiDr cells was initially performed under an inverted microscope (Fig 2). As seen in Fig 2a cell were slightly more rounded in shape and in mitotic phase than other. Cell incubated with cinna- mon oil 500 µg/ml for 24 hr showed obvious changes in their morphology such as the loss of adhesion, elongated and rounded in shape. Effect of the cinnamon oil on the morphology and number of WiDr cells stonger and dose dependent. These morphological observations were consistent with the results of the cytotox- icity and apoptosis experiment.

  Doxorubicin generally require p53 to induce apoptosis (Drummond, 2007). Protein p53 is a tumor suppressor gene involved in apoptosis pacing. WiDr cells are cells with mu- tated p53 thus allowing cinnamon oils are able to induce apoptosis through p53 - independent pathway. The use of doxorubicin may cause the activation of the transcription factor NF- kB. The presence of NFkB activation can lead to activation of antiapoptosis proteins such as Bcl - xl (Pahl, 1999). With the inhibition of the activation of NFkB activation anti apoptosis proteins can also be inhibited so that the pro- cess of apoptosis can occur. However, more re- search is needed on the effect of cinnamon oil, alone or in combination on the expression of proteins involved in the modulation of apop- totic pathways in WiDr cells to determine the mechanism.

  Figure 3. Curve comparison of the log-dose relationship versus percent living Vero cells (cell viability) with cinnamon oil concentrations of 3,9-500 µg/ml.

  Cytotoxic effect of cinnamon oil to Vero cells was show on figure 3. Figure 3 curve com- parison of the log-dose relationship versus percent living Vero cells (cell viability) with cinnamon oil concentrations of 3,9-500 µg/ml. The IC value of cinnamon oil on Vero culture cells was 27,35 µg/ml.

  It was our main observation that growth of both cell lines was inhibited in a concentra- tion-dependent manner to cinnamon oil. The

  IC50 value of cinnamon Oil on WiDr cell lines was 38,02 µg/ml and 27,35 µg/ml. According to Ueda et al

  . (2002) extracts which have IC50 values below 100μg/ml have potent cytotoxic effects. These result indicate that some con- stituens of the oil may interfere with ras trans- formation (Zeytinoglu et al., 2003). The potent cytotoxic activity showed that the cinnamon oil potential to be developed as a chemopre- ventive agent in the treatment of cancer, espe- cially colon cancer.

  The cytotoxic properties of various es- sential oils and their constituents linked to an anticarcinogenic activity. It is well document- ed that many monoterpens from essential oils such as limonene and geraniol inhibit isopre- nylation of proteins (Crowell, 1999; Gelb et

  al

  ., 1995). More over ras-oncogene induced carcinomas have been prevented by limonene, geraniol and 2’-benzyloxy cinnamaldehyde which is a constituent of Cinnamomum oil (Moon et al., 2006; Carnesecchi et al., 2004; Gould et al

  ., 1994). The prenylation of Ras en- ables it to associate with plasma membrane, which is required for its oncogene activity Ras is a small gunosie tri phosphatase (GTPase) with a signal-transducing functon that plays key roles in the control of cells growth and differ- entiation (Marshall, 1991). Therefore, impair- ment of the prenylation of Ras might account for the antitumor activity of some componens of the essential oil. Previous research proved that sinamaldehid, one of the active compound content in cinnamon, able to induce apoptosis

  W Herdwiani, F Leviana, Z Imama, Sari, AA Soemardji, Elfahmi, MI Tan

  CYTOTOXICITY EFFECT OF CINNAMON OIL ON WiDr & Vero CULTURE CELL AND SUB CRONIC

TOXICITY EFFECT OF CINNAMON OIL ON ALT/AST LEVELS MALE RAT

  Efek Sitotoksisitas Minyak Kayu Manis Terhadap Kultur Sel WiDr & Vero Serta Efek Toksisitas Subkronis Minyak Kayu Manis Pada Kadar ALT/ASL Tikus Putih Jantan

  Tabel 3. The results of the analysis of the average levels of

  through the mitochondrial permeability tran-

  SGPT / ALT white male rats

  sition induced apoptosis (MPT) (Ka et al.,

  Group Average of SGPT/ALT levels (U/L)

  2003). Cytotoxic activity of the essential oil

   (t0) (t1) (t2) (t3) (t4)

  was initially thought to be caused because the

  Aquadest 25,6 26,92 26,94 26,38 26,34

  Dose I

  active compounds contained in cinnamon is si-

  23,8 23,14 25 27,5 25,94 Dose II

  24,8 26 24,52 26,48 27,22

  namaldehid. However, based on the results of

  Dose III 27,2 29,86 28,06 30,6 32,04

  the CCRC unpublished data (2012), pure sina-

  50

  maldehid showed IC values greater than the distillate cinnamon (MGD) or in other words sinamaldehid less potent than MGD. Based on the study Singh et al

  . (2009) aqueous cinna- mon extract/ACE showed cytotoxic effect at a concentration of 0,16 mg/ml (containing 1,28 μM sinamaldehid). More potent cytotoxic activ- ity on ACE. ACE is due to the polyphenols con- tained other components that have synergistic activity with sinamaldehid. Further research is

  Figure 4. The grafics of Groups Vs SGPT levels

  needed to determine what components of the essential oil of cinnamon bark that play a role The result of SGOT/AST assay in the cytotoxic activity both on WiDr and Vero

  Table 4. The result of SGOT /AST assay culture cells.

  Group The Average of SGOT assay (U/L) (t0) (t1) (t2) (t3) (t4) Toxicity sub chronic effect of Cinnamon Oil

  Aquades t 110,8 111,4 113,8 112,4 111,8

  Dose I 111,2 112,2 108,2 111,8 114,2

  Table 2 was a result of weight body of

  Dose II 109 117,2 115,4 117,4 121

  the animal trial. Based on the SPSS of body

  Dose III 114,6 119,8 120,6 124,2 125,6

  weight result that wasn’t siqnificant difference on all groups.

  Table 2 . The results of the analysis of the average weight of male mice Group The Average of Body Weight (gram) (t0) (t1) (t2) (t3) (t4) (-) Control

  199,4 199,8 199,8 200,2 200,4 Dose I

  200 200,6 199,8 200,2 200,6 Dose II

  201 201,4 200,8 201 201,2 Dose III

  200,8 201,6 200,6 200,6 202,2 Figure 5. The grafics of Group Vs SGOT/AST (U/L)

  The result of SGPT/ALT assay was The result of SGOT/AST assay was showed at Table 3 and Fig 4 were viewed chart showed at Table 4 and Fig 5 were viewed chart

  Groups Vs SGPT levels. Based on SPSS result Groups Vs SGOT levels. Based on SPSS result from this SGPT levels showed that there was from this SGOT levels showed that there was not siqnificant difference in all groups. not siqnificant difference in all groups.

  The result of microskopic assay of hepar was showed at Figure 6. Figure 7 was viewed result of microscopic aboservation on the sam- ple. W Herdwiani, F Leviana, Z Imama, Sari, AA Soemardji, Elfahmi, MI Tan

  has a toxicity activity to while male rat on hep- ar.

  ACKNOWLEDGMENTS

  We are thankful to the Indonesian Min- istry Of Education for funding this research. Prof. Dr. Zullies Ikawati, Apt and Dr Triana Hertianti, Apt. for helping in this research.

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