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Volume 1, Nomor 1, Februari 2011

Volume 1, Nomor 1, Februari 2011 ISSN : 2087-5045 S S c c i i e

ISSN : 2087-5045

Volume 1, Nomor 1, Februari 2011 ISSN : 2087-5045 S S c c i i e
Volume 1, Nomor 1, Februari 2011 ISSN : 2087-5045 S S c c i i e
Volume 1, Nomor 1, Februari 2011 ISSN : 2087-5045 S S c c i i e

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Penanggung Jawab :

Prof. H. Syahriar Harun, Apt

Pemimpin Umum :

DR.H.M. Husni Mukhtar,MS, DEA, Apt

Redaktur Pelaksana :

Verawati, M.Farm, Apt Eka Fitrianda, M.Farm, Apt

Sekretariat :

Afdhil Arel, S.Farm, Apt Khairul

Dewan Penyunting :

Prof.H. Syahriar Harun,Apt Prof.DR.H. Amri Bakhtiar,MS,DESS,Apt Prof.DR.H. Almahdy, MS, Apt DR.H.M. Husni Mukhtar, MS, DEA, Apt DR.Hj. Marlina, MS, Apt Drs. Yufri Aldi, MSi, Apt Drs. B.A. Martinus , MSi Hj. Fifi Harmely, M.Farm ,Apt Farida Rahim, M.Farm, Apt Revi Yenti, M.Si, Apt Verawati, M.Farm, Apt Ria Afrianti, M.Farm ,Apt Eka Fitrianda, M.Farm, Apt

Penerbit :

Sekolah Tinggi Farmasi Indonesia (STIFI) Perintis Padang

ISSN : 2087-5045

Alamat Redaksi/Tata Usaha STIFI Perintis Jl. Adinegoro Km. 17 Simp. Kalumpang Lubuk Buaya Padang Telp. (0751)482171, Fax. (0751)484522

e-mail : stifi_perintis@yahoo.co.id website : www.stifi-padang.ac.id

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ANALISA KANDUNGAN FLAVONOID DAN AKTIVITAS ANTIOKSIDAN DARI REMPAH TUMBUHAN OBAT SUMATERA BARAT

Deddi Prima Putra 1 , Verawati 2 1 Fak. Farmasi Universitas Andalas, 2 STIFI Perintis Padang

ABSTRACT

Antioxidant activity and total flavonoid content of five medicinal plant specieses of West Sumatera have been measured. The spesieses are Jahe (Zingiber officinale Rosc), Kunyit (Curcuma domestica Val), Kencur (Kaemferia galanga Linn), Lengkuas (Alpinia Galanga Linn), and Pala (Myristica fragrans Houtt). The total flavonoid content and antioxidant activity were measured on total ethanolic extract, liphofilic fraction and hydrophilic fraction of each species. The total flavonoid content was measured using colorimetric with alumunium chloride used as complexing agent and quercetin used as standard compound.The examination of antioxidant activity was carried out by spectrophotometry UV-Visible using DPPH reagent. The total flavonoid content of Jahe (Zingiber officinale Rosc), Pala (Myristica fragrans Houut), Kunyit (Curcuma domestica Val) were 0.85; 0.54; 19.77 µg/g respectively (quercetin equivalent).The result showed that these three plants have highest antioxidant activity. IC 50 of jahe against 20 µg/ml DPPH were 80.62, 69.35, 109.98 µg/ml for total ethanolic extract, liphofilic fraction and hydrophilic fraction respectively. IC 50 of ethanolic extract and hydrophilic fraction of kunyit were 68.21 and 47.09 µg/ml, while IC 50 of ethanolic extract and hydrophilic fraction of pala were 50.08 and 71.67 µg/ml.

Keywords : Antioxidant, Flavonoid, medicinal plants

PENDAHULUAN

Obat asli Indonesia merupakan obat yang berasal dari tumbuhan, hewan, atau bahan mineral. Pada umumnya obat asli Indonesia belum mempunyai data klinik dan penggunaannya hanya berdasarkan pengalaman. Pengolahan obat asli Indonesia masih sederhana dengan menyeduh bahan tumbuhan kering atau segar dengan air panas, kemudian air seduhan ini diminum. Oleh karena itu bahan obat asli Indonesia perlu

distandarisasi, sehingga manfaat dan

dapat

keamanannya

dipertanggungjawabkan. Dengan demikian tumbuhan obat asli Indonesia dapat dikembangkan menjadi fitofarmaka (Depkes, 1981).

Rempah-rempah sudah lama dikenal di Indonesia. Makanan sehari- hari kita mengandung paling tidak satu jenis rempah. Rempah punya arti lebih dari sekedar penambah rasa hidangan. Rempah tumbuhan obat juga berpotensi besar memerangi sederet panjang penyakit dan masalah kesehatan seperti kanker, jantung, diabetes melitus, dan arterosklerosis. Pemicu timbulnya penyakit-penyakit tersebut salah satunya adalah akibat radikal bebas (Rungkat,

1994).

Radikal bebas adalah senyawa kimia yang mempunyai satu atau lebih elektron tidak berpasangan. Senyawa ini bersifat tidak stabil dan sangat reaktif. Untuk mencapai kestabilan, molekul harus mencari elektron lain sebagai pasangan. Reaksi berantai ini dapat menimbulkan kerusakan sel yang berujung pada mutasi sel dan apabila

1

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merusak organ yang memiliki fungsi

alumunium

klorida

sebagai

tertentu dapat menimbulkan penyakit

pengompleks

dan

penentuan

aktivitas

degeneratif. Radikal bebas dalam

antioksidan

menggunakan

metode

kehidupan sehari-hari dapat dijumpai

radikal

DPPH

(Zhinshen,

1999;

seperti akibat metabolisme yang

Molyneux, 2004).

 

berlebihan atau berasal dari lingkungan seperti asap rokok, polusi udara, bahan

METODE PENELITIAN

 

kimia beracun, pestisida serta radiasi sinar UV (Raharjo, 1992; Silalahi, 2001;

Alat dan Bahan

 

Youngson, 2005).

Alat

Untuk menanggulangi efek dari radikal bebas ini, secara alami tubuh mempunyai benteng yang dapat mencegah serangan radikal bebas tersebut yaitu enzim (katalase) ataupun antioksidan yang berasal dari luar tubuh yang umumnya berasal dari makanan. Kegunaan utama dari antioksidan adalah menghentikan atau memutuskan reaksi berantai dari radikal bebas dengan cara menyediakan dirinya bereaksi dengan radikal bebas itu sendiri. Dengan kata lain, antioksidan dapat menyelamatkan sel-sel tubuh dari kerusakan, akibat serangan radikal bebas (Karyadi,1997).

Antioksidan dapat berasal dari alam maupun sintetik. Antioksidan alam lebih disukai karena efek sampingnya lebih kecil. Salah satu sumber antioksidan alam yang terdapat dalam tanaman adalah flavonoid yang bisa ditemukan pada beberapa tanaman dari Famili Zingiberaceae seperti rimpang kunyit (Curcuma domestica. Val), rimpang kencur (Kaemferia galanga. Linn), rimpang jahe (Zingiber officinale. Rosc), rimpang lengkuas (Alpinia galang. Linn, Willd), dan dari tanaman famili Myristicaceae seperti buah Pala (Myristica fragrans. Houtt) (Rukmana. 1994; Rukmana, 1995).

Berdasarkan potensi rempah tersebut sebagai obat dan adanya kandungan flavonoid didalamnya, maka dilakukan penelitian untuk menentukan kadar flavonoid total dalam rempah secara kolorimetri dengan menggunakan

Alat yang digunakan berupa seperangkat alat maserasi, rotary Evaporator (BUCHI ® ), labu rotary, Erlemeyer berbagai ukuran, corong, kapas, pipet tetes, pipet mikro, botol, vial, label, spatel, alumunium foil, timbangan digital, spektrofotometri UV- Vis Pharmaspec 1700 (shimadzu ® ), mesin penghalus (Brook Crompton ® ), pisau, tabung reaksi, rak tabung reaksi.

Bahan

Sampel rempah-rempah:

rimpang kunyit (Curcuma domestica. Val), rimpang lengkuas (Alpinia galanga. Linn, Willd), rimpang jahe (Zingiber officinal., Rosc), rimpang kencur (Kaemferia galanga. Linn) dan buah pala (Myristica fragrans. Houtt), yang masing-masing diambil dari daerah berbeda yaitu Alahan Panjang Bukittinggi dan Batu Sangkar, etanol 96%, heksan 96%, metanol, DPPH, Natrium asetat 1 M, AlCl 3 10%, Aquadest, Quersetin, Aquadest.

Pembuatan Ekstrak Sampel

Serbuk sampel sebanyak 5 g diekstraksi dengan menggunakan pelarut yang berbeda sehingga diperoleh 3 macam ekstrak.

a. Pembuatan ekstrak etanol total (ekstrak total): 5 gram sampel diekstrak dengan 25 ml etanol 96% selama 2 x 24 jam. Kemudian

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b. maserat disaring dan filtrat dikentalkan dengan rotary evaporator sehingga diperoleh ekstrak kental total.

sedangkan IC 50 sampel dihitung dengan metoda analisa probit menggunakan finney.

 

Penentuan

Kandungan

Flavonoid

c. Pembuatan ekstrak heksan (ekstrak

d. Pembuatan ekstrak etanol setelah

Total

lipofil): 5 gram sampel diekstrak dengan 25 ml heksan 96% selama 2 x 24 jam. Kemudian maserat disaring dan filtrat dikentalkan dengan rotary evaporator sehingga diperoleh ekstrak kental lipofil.

heksan (ekstrak hidrofil): ampas dari hasil ekstraksi dengan heksan diekstraksi lagi dengan etanol 96% sebanyak 25 ml selama 2 x 24 jam. Kemudian maserat disaring dan filtrat dikentalkan dengan rotary evaporator sehingga diperoleh ekstrak kental hidrofil.

Kandungan flavonoid total ditentukan dengan metode kolorimetri menggunakan Aluminium klorida. Sebanyak 2 ml dari larutan ekstrak (1mg/ml) serta larutan standar kuersetin (100; 80; 60; 40; 20; 10; 5 µg/ml) ditambah 0,1 ml AlCl3 10 %; 0,1 ml Na asetat 1M dan 2,8 ml air suling. Campuran dikocok homogen lalu biarkan selama 30 menit. Serapan diukur pada panjang gelombang 415 nm. Total kandungan flavonoid sampel dinyatakan sebagai kesetaraan gram kuersetin/100 gram sampel kering.

HASIL DAN PEMBAHASAN

Penentuan

Aktivitas

Antioksidan

Sampel

Setelah dilakukan penelitian

Aktivitas antioksidan ditentukan dengan metode DPPH (Molyneux, 2004). 0,2 ml larutan sampel (1 mg/ml, 100, 50 dan 25 µg/ml) serta larutan stándar kuersetin (0,1; 0,2; 0,4; 0,6 g/ml) di dalam vial, ditambah 3,8 ml larutan DPPH (20 µg/ml). Campuran larutan dihomogenkan dan dibiarkan selama 30 menit di tempat gelap. Serapan diukur pada panjang gelombang maksimum DPPH yaitu 517 nm. Absorban kontrol yaitu DPPH (20 µg/ml) dalam metanol juga diukur.

mengenai analisa kandungan flavonoid total dan aktivitas antioksidan dari tanaman obat dan rempah Sumatera Barat didapatkan hasil bahwa dari 5 macam sampel rempah (jahe, kunyit, kencur, lengkuas dan pala) didapatkan tiga sampel memiliki aktivitas antioksidan yang tinggi yaitu jahe, kunyit dan pala. Ketiga rempah ini ditentukan IC 50 nya yakni konsentrasi sampel yang mampu merangkap radikal DPPH sebesar 50%. Semakin kecil nilai IC 50 maka semakin aktif sampel tersebut sebagai antioksidan.

Aktivitas antioksidan sampel dinyatakan sebagai persentase inhibisi dihitung dengan rumus:

Abs. kontrol – Abs. Sampel

Abs. kontrol

x 100 %

Dengan menggunakan persamaan linear dari data stándar maka dapat dihitung IC 50 stándar kuersetin

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Tabel I. Nilai IC 50 sampel

No

Nama sample

Konsentrasi

% Inhibisi

IC 50

   

100

52.96

 

1

Jahe.BS.Total

50

36.14

90.56

25

21.51

 

100

69.31

 

Jahe.BS.Lipofil

50

49.24

57.67

25

32.41

 

100

69.89

 

Jahe.Bkt.Total

50

47.61

60.68

25

29.06

 

100

63.38

 

Jahe.Bkt.Lipofil

50

44.93

68.98

25

26.58

 

100

58.63

 

Jahe.Bkt.Hidrofil

50

39.45

79.3

25

22.54

 

100

52.81

 

Jahe.Ap.Total

50

36.59

90.62

25

21.95

 

100

57.22

 

Jahe.Ap.Lipofil

50

39.12

81.4

25

23.08

 

100

35.54

 

Jahe.Ap.Hidrofil

50

17.50

140.66

25

8.83

   

100

50.72

 

2

Kunyit.BS.Total

50

28.18

97.92

25

15.09

 

100

92.58

 

Kunyit.BS.Hidrofil

50

58.26

46.81

25

28.04

 

100

89.59

 

Kunyit.Bkt.Total

50

65.76

32.44

25

42.12

 

100

85.9

 

Kunyit.Bkt.Hidrofil

50

59.09

46.51

25

30.27

 

100

60.85

 

Kunyit.Ap.Total

50

43.82

74.27

25

21.48

 

100

73.09

 

Kunyit.Ap.Hidrofil

50

49.72

47.97

25

25.95

   

100

78.36

 

3

Pala.BS.total

50

49.72

54.44

25

29.4

 

100

76.18

 

Pala.BS.Hidrofil

50

43.38

61.55

25

23.72

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100

88.47

 

Pala.Bkt.Total

50

57.56

45.69

25

31.85

 

100

58.81

 

Pala.Bkt.Hidrofil

50

36.09

81.8

25

16.45

Keterangan : Ap : Alahan Panjang BS : Batu Sangkar Bkt : Bukittinggi

Dari data IC 50 sampel dan IC 50 stándar kuersetin, maka diperoleh suatu nilai jumlah sampel yang akan memberikan aktivitas antioksidan yang setara dengan 1 mg kuersetin (IC 50 kuersetin = 0,442 µg/ml).

Tabel II. Aktivitas Antioksidan dari Sampel Setara mg Kuersetin

No

Nama sampel

Nilai rata-rata IC 50 aktivitas antioksidan terukur (µg/ml)

Aktivitas antioksidan 1g ekstrak setara mg kuersetin (mg)

 

Jahe. total

80.62

182.39

1

Jahe. lipofil

69.35

156.9

Jahe. hidrofil

109.98

248.82

 

Kunyit. total

68.21

154.32

2

Kunyit. lipofil

-

-

Kunyit hidrofil

47.09

106.53

 

Pala. total

50.06

113.26

3

Pala. lipofil

-

-

Pala. hidrofil

71.67

162.16

Pada pengukuran absorban flavonoid total untuk penentuan kurva kalibrasi kuersetin pada panjang gelombang 415 nm didapat persamaan regresi y = - 0,016 + 0,017x dengan koefisien korelasi 0,999, simpangan baku 0,0106371, batas deteksi – 0,114942 g/ml, batas kuantisasi 4,216 g/ml.

Pada pengukuran flavonoid total tiap gram sampel kering setara kuersetin diperoleh kadar flavonoid total dari masing-masing sampel dimana kadar paling tinggi adalah pada ekstrak etanol kunyit 19.70 µg/g diikuti kencur 0.92 µg/g, jahe 0.84 µg/g, pala 0.54 µg/g, lengkuas 0.52 µg/g.

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Tabel III. Kandungan Flavonoid total dari ekstrak total sampel

Nama Sampel

Konsentrasi Flavonid (µg/ml)

Kadar Flavonoid tiap 5 g serbuk kering (µg/g)

Jahe.BS.Total

10.62

0.91

Jahe.Bkt.Total

 

8.1

0.70

Jahe.AP.Total

10.83

0.93

Rata-rata ± SD

9.85

± 1,51

0.84

± 0,13

Kunyit.BS.Total

157.55

25.03

Kunyit.Bkt.Total

71.99

11.43

Kunyit.AP.Total

142.67

22.66

Rata-rata ± SD

124.07 ± 45,71

19.70 ± 7,26

Kencur.BS.Total

 

8.5

0.84

Kencur.Bkt.Total

 

8.64

0.85

Kencur.AP.Total

11.03

1.09

Rata-rata ± SD

9.39

± 1,42

0.92

± 0,14

Lengkuas.BS.Total

4.53

0.37

Lengkuas.Bkt.Total

9.27

0.76

Lengkuas.AP.Total

 

5.02

0.41

Rata-rata ± SD

6.27

± 2,60

0.52

± 0,21

Pala.BS.Total

4.96

0.60

Pala.Bkt.Total

4.05

0.49

Rata-rata ± SD

4.50

± 0,64

0.54 ± 0,078

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Dari data IC 50 , aktivitas antioksidan dan penentuan kadar flavonoid total diketahui bahwa ekstrak yang kadar flavonoidnya tinggi mempunyai aktivitas antioksidan yang tinggi. Jadi kemungkinan senyawa yang mempunyai aktivitas antioksidan yang tinggi adalah golongan flavonoid yang terdapat pada ekstrak polar atau fraksi hidrofil terutama pada jahe, kunyit dan pala.

KESIMPULAN

Berdasarkan hasil penelitian yang telah dilakukan dapat diambil kesimpulan bahwa dari 5 tanaman yang berasal dari 3 daerah, kadar flavonoid yang tinggi terdapat pada kunyit, jahe dan pala setara kuersetin berturut-turut :

19.77; 0.85; 0.54 g/g terhadap sampel kering. Ekstrak hidrofilik memberikan aktivitas antioksidan lebih tinggi dibandingkan ekstrak lipofilik.

DAFTAR PUSTAKA

Direktorat Jenderal Pengawasan Obat dan Makanan Departemen Kesehatan RI, 1981, Modifikasi Peraturan Perundang – undangan Obat Tradisiona, Jakarta. Karyadi, E., Antioksidan, Resep Sehat dan Umur Panjang, http//www.indomedia.com/intisari

/1997/juni/antioksidan.htm

Molyneux, P., 2004, The Use of Stable Free Radical Diphenyl- picrylhydrazyl (DPPH) for Estimating Antioxidant Activity, J. Sci. Tecnol, 26(2), 211-219. Raharjo, M., 1992, Tanaman Berkhasiat Antioksidan, Penebar Swadaya, Jakarta. Rukmana, R., 1995, Temulawak Tanaman Rempah Obat, Kanisius, Yogyakarta.

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Rukmana, R., 1994, Kencur, Kanisius, Yogyakarta. Rungkat, F., 1994, Radikal Bebas dan Patofisiologi Penyakit, Proseding Seminar : Senyawa Radikal dalam Sistem Pangan : Reaksi Biomolekuler, Dampak terhadap Kesehatan dan Penangkalan, Bogor.

Silalahi, J., 2001, Free Radicals and antioxidant Vitamin in Degenerative Disease. The Journal of Indonesian Medical Association : II (5), 1-13. Youngson, R., 2005, Antioxidant :

Vitamin C dan E For Health, diterjemahkan oleh Susi Purwoko, Jakarta. Zhinshen, J., T. Mengcheng and W. Jianming, 1999, The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals, Food Chem., 64: 555-

559.

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ISOLATION OF Vibrio parahaemolyticus FROM BEEF MARKETED IN PADANG, IDENTIFICATION TARGETED ON toxR GENE AND AMPLIFICATION OF tdh AND trh GENES ON ISOLATES USING POLIMERASE CHAIN REACTION

Eka Fitrianda 1 , Marlina 2 , M. Husni Mukhtar 2 1 STIFI Perintis Padang, 2 Fakultas Farmasi Universitas Andalas Padang

ABSTRAK

Sebanyak 40 isolat V. parahaemolitycus telah berhasil diisolasi dari sejumlah sampel daging sapi mentah yang dikoleksi di Pasar Raya Padang. Isolasi dilakukan dengan menggunakan media CHROMagar TM Vibrio. Terhadap 40 isolat tersebut dilakukan identifikasi menggunakan metoda Polymerase Chain Reaction (PCR) untuk mengamplifikasi gen toxR. Gen toxR merupakan gen yang sangat spesifik pada spesies V. parahemolyticus. Selanjutnya, dengan metoda yang sama juga dilakukan amplifikasi terhadap gen pengkode produksi toksin hemolisin (tdh dan trh) yang merupakan faktor virulen utama pada bakteri tersebut. Hasil penelitian menunjukkan bahwa seluruh V. parahaemolyticus hasil isolasi memiliki gen tox-R, namun tidak ada satu isolatpun yang memiliki gen pengkode produksi toksin hemolisin baik tdh maupun trh.

Key words: Vibrio parahaemolyticus, toxR, tdh, trh

INTRODUCTION

V. parahaemolyticus is one of bacteria actively studied in various parts of the world because of its ability in causing diarrhea. V. parahaemolyticus was first isolated in food poisoning outbreak in Japan in early 1950s. Currently, V. parahaemolyticus has become one of food contaminant pathogens with the highest prevalence in Asian countries (Pan et al, 1997). The main clinical manifestation of infection due to V. parahaemolyticus is gastroenteritis, in which the main symptoms include abdominal cramps, nausea and vomiting.

Most strains of clinical V. parahaemolyticus produce major virulence factor namely thermostable direct hemolysin (TDH) and showed β- hemolysis activity on Wagatsuma agar (KP positive strains). Another virulence factor, TDH-related hemolysin (TRH), usually associated with KP negative strains or with urease positive strains

8

(Kelly and Stroh, 1989).

Based on the Shirai et al (1990) report, molecular epidemiological studies show

a close relationship between the

hemolysin coding genes in these bacteria (tdh, trh, or both) with their ability in causing disease. Thermostable direct hemolysin (TDH) and the TDH-

related hemolysin (TRH), whose production is encoded by the tdh and trh

are the important virulence factors for

the development of gastroenteritis.

Therefore, the genes are referred to as

the important virulence coding genes in

V. parahaemolyticus.

Although V. parahaemolyticus is a marine bacterium that has long been associated with diarrhea after eating raw or not cooked perfectly seafood, some recent researches showed that V. parahaemolyticus has also found

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on food samples which are not seafood

Salt

Polimixin

Broth

(SPB),

distilled

categories. Research conducted by

water,

NaCl,

CHROMagar TM

Vibrio

Marlina et al (2007) for example, has

(CHROMagar TM ),

Luria

Bertani

(LB)

successfully isolated these bacteria from

Broth, Luria Bertani (LB), 5X Colorless

pensi (Corbicula moltkiana prime) collected from lake Singkarak. The

GoTaq Reaction, 10X Ex Taq buffer solution, 2.5 mM dNTP solution, GoTaq

isolated strains carried hemolysin toxin- producing genes (tdh and trh) which are the major virulence factors of V.

DNA polymerase, agarose, tris-borate- EDTA (TBE), a blue dye, 100 bp ladder, ethidium bromide, and three pairs of

parahaemolyticus. Another study

primers,

namely:

conducted by Zulkifli et al (2009) on the

toxR-4:

5'-

cockles collected from rivers in Padang

GTCTTCTGACGCAATCGTTG-3' and

showed similar result.

toxR-7:

5'-

ATACGAGTGGTTGCTGTCATG-3'

In

this

study

we

isolated

V.

for

the

detection

of

toxR

gene,

parahaemolyticus from beef samples

MATERIALS AND METHODS

TDH

D3:

5'-

and identified them by examining on

CCACTACCACTCTCATATGC-3’ and

their toxR gene. We also detected their

TDH

D5:

5'-

virulent factors coding genes by

GGTACTAAATGGCTGACATC-3' for

amplificating tdh and trh genes on these

detection

of

tdh

gene,

isolates.

TRH

R2:

5'-

The method used to identifying and

GGCTCAAAATGGTTAAGCG-3'

and

amplifying tox-R, tdh and trh genes is

TRH

R6:

5'-

polymerase chain reaction (PCR). PCR

CATTTCCGCTCTCATATGC-3'

for

is a major breakthrough in molecular

detection

of

trh

gene.

diagnostics, and lately has been widely used to identify genes from different

Sampling

 

species of bacteria including V. parahaemolyticus. According to Gelfand et al (1998), this is due to the high sensitivity level of this method.

The test samples in this study were 15 raw beef samples. Samples were taken from traders in Pasar Raya Padang. To avoid contamination, samples were taken in a way directly

V. parahaemolyticus Isolation

Equipments and materials

 

entered by the merchant into sterile containers, immediately stored in

PCR machine (Eppendorf Mastercyclergradient ® ), Eppendorf tubes, micro pipette (Eppendorf ® ),

containers and taken to the laboratory for testing.

centrifugator (Eppendorf Minispin ® ), laminar air flow (Esco ® ), vortex (Mixer ® VM-1000), incubator (Gallenkamp ® ), rotary shaker incubator (Bigger Bill Digital ® ), colony counter (Stuart scientific ® ), the elektrophoresis device, transilluminator, polaroid film, the test sample, V. Parahaemolyticus AQ4037 (positive control for the toxR and trh genes), V. parahaemolyticus AQ3815 (positive control for tdh gene,),

Each sample (10 gram) was added in to 100 ml Salt Polimixin Broth (SPB) media and incubated at 37°C for 24 hours. These cultures were inoculated using a needle loop onto surfaces of CHROMagar TM Vibrio previously been poured and allowed to solidify in a petri dish. After incubated for 24 hours at

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37°C, purple colonies formed were suspected as colonies of V. parahaemolyticus. Each single suspected colony was inoculated into the Luria Bertani (LB) Broth containing 3% NaCl and incubated in a rotary shaker incubator at 37°C for 24 hours.

DNA template preparation

Before the amplification of genes using PCR method, DNA of control and testing bacteria were extracted using boil cell extraction (BCE) method. 1 ml LB broth culture centrifuged at 12,000 rpm for 1 min, the supernatant were removed, while the sediment were added with 500 mL sterile distilled water and then suspended using vortex. The suspension formed was heated for 10 minutes in boiling water and immediately put into the refrigerator with a temperature of 20ºC for 10 minutes. Subsequently centrifuged at 12,000 rpm for 3 minutes and the supernatant were transferred into a new Eppendorf tube. The supernatant were the DNA template to be used for amplification of genes by PCR method.

Amplification

of

toxR,

tdh

and

trh

genes

Identification of toxR, tdh and trh genes in V. parahaemolyticus were done using PCR method. DNA template which had been prepared previously inserted into the Eppendorf tube for PCR machine and combined with other PCR components. The type and amount of each PCR components as shown in table below:

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Table I. PCR component

Component

Amount(μl)

Buffer solution a

5,0 c 2,5 d

2,5 mM dNTP

2,0

Primer 1 b

1,0

Primer 2 b

1,0

Aquadest

15,0 c 17,4 d

GoTaq DNA Polymerase

0,1

DNA Template

1,0

a 5x Colorless GoTaq Reaction Buffer for the amplification of tdh and trh genes, 10x Ex Taq buffer solution for the amplification of toxR gene, b Primer pairs are suitable for each gene, c For the detection of tdh and trh genes, d For the detection of toxR gene

Eppendorf tube is then inserted into the PCR machine and amplification was performed using program which is suitable for detection of each gene as shown in table below:

Table

II.

Stage

for

tox-R

gene

amplification (23 cycles)

 

Temperature ( o C )

Time

Stage

(minute)

Predenaturation

96

5

Denaturation

94

1

Annealing

63

1,5

Extention

72

1,5

Elongation

72

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Table III. Stage for tdh and trh genes amplification (33 cycles)

Stage Predenaturation Denaturation Annealing Extention Elongation Temperatur e ( o C ) 96 94 55

Stage

Stage Predenaturation Denaturation Annealing Extention Elongation Temperatur e ( o C ) 96 94 55 72

Predenaturation

Denaturation

Annealing

Extention

Elongation

Temperatur

e

( o C )

96

94

55

72

72

Time

(minute)

5

1

1

2

7

After all of the stages in the PCR process were completed, the results

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of this amplification were colored using blue dye and then separated along electrophoresis process on 1% agarose gel in 1x TBE. Electrophoresis was performed at 100 V voltage for 20 minutes use 1x TBE as the mobile phase. The 100 bp ladder was used as a marker for determining the size of amplification product. After the electrophoresis process was completed, agarose gel was stained using ethidium bromide solution (0.5 mL/ml). Electrophoresis result which was observed by UV transilluminator will form separate bands which were distinguished by the number of their base pairs (bp). The size of toxR, tdh and trh amplification product were 368 bp, 251 bp and 250 bp respectively. Size estimation of each product was done through comparison with 100 bp ladder. Results were then documented by polaroid film.

RESULTS AND DISCUSSION

We successfully isolated suspected V. parahaemolyticus colonies from 3 of 15 raw beef samples which were examined. The presence of suspected V. parahaemolyticus in samples characterized by the formation of purple colonies on the surface of CHROMAgar TM Vibrio medium.

colonies on the surface of CHROMAgar T M Vibrio medium. Figure 1. Suspected V. parahaemolyticus colonies

Figure 1. Suspected V. parahaemolyticus colonies on Chromagar TM Vibrio.

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CHROMagar ™ Vibrio is a selective media for identification of V. parahaemolyticus with a higher level of differentiation compared to TCBS medium (Kudo etal, 2001). From 3 of 15 samples which were examined, we successfully isolated 40 suspected V. parahaemolyticus. Identification targeted on toxR gene was performed to all of these isolates using PCR method. toxR gene is a gene that is very specific on the V. parahemolyticus species. The PCR method to detect this gene has been reported (Lee et al, 1995) as a very useful method to confirm the presence of this species in samples. Dileep et al (2003) also states that the detection of toxR gene by PCR method to detect V. parahaemolyticus is more sensitive than biochemical identification.

toxR positive isolates showed

368 bp band in electrophoresis gel. In this study, out of total of 40 tested

isolates,

toxR

positive results.

all

(100%)

showed

40 tested isolates, toxR positive results. all (100%) showed 1 2 3 4 5 6 7

1 2

3

4

5

6

7

8

9

10 11 12 13

Figure 2. Electrophoresis gel of toxR positive V. parahaemolyticus isolates: lane 1-11 were positive toxR isolates, lane 12 was positive control, lane 13 was 100 bp ladder.

The same result have been reported by Zulqifli et al (2009), where all of CHROMagar™ Vibrio isolates from cockles gave positive results on testing of toxR using PCR method.

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Previously, V. parahaemolyticus has been isolated from various places around the world. Tanil et al (2005) succeeded in isolating V. parahaemolyticus from seawater in Peninsular, Malaysia. V. parahaemolyticus also been isolated from coastal waters of western United States (Okuda et al, 1997). A study recently conducted by Sujeewa et al (2009) succeeded in isolating V. parahaemolyticus from shrimp samples in Malaysia. Generally, V. parahaemolyticus were isolated from marine waters or food samples from the sea, because V. parahaemolyticus is a bacteria that normally lives in this habitat (DePaola et al, 2000).

It makes the results of this research is interesting for further explored, because V. parahaemolyticus isolates were isolated in samples which were not derived from the sea environment. Similar results have also found in other study (Marlina et al, 2007), where a number of V. parahaemolyticus isolates carrying tdh gene were isolated from Corbicula moltkiana, a species which lives in lake Singkarak. Similarly, other publication also showed that V. parahaemolyticus was isolated from cockles live in rivers around Padang, Indonesia (Zulkifli et al, 2009). Furthermore, studies are necessary to investigate whether the presence of V. parahaemolyticus in samples is the result of bacterial adaptation capabilities on low-salt environment, or is the result of cross- contamination when samples are marketed in the market.

Major virulence factor coding genes in V. parahaemolyticus are tdh and trh. The presence of these genes are represent the level of pathogenicity of V. parahaemolyticus isolates. In this study, 40 toxR positive isolates were detected for the present of tdh and trh genes

12

using PCR method. As a result, none of the isolates has tdh or trh gene. This result suggests that V. parahaemolyticus isolates in this study are not virulent isolates. According to Nishibuchi and Kaper (1995), only few V. parahaemolyticus isolates carrying the virulent genes (tdh or trh), and they are called virulent isolates. The existences of these genes in V. parahaemolyticus isolated from nonclinical samples are rarely detected. In contrast, more than 90% of V. parahaemolyticus isolates from clinical samples have tdh or trh gene (DePaola et al, 2000). These such result also seen in V. parahaemolyticus previously isolated from cockle samples in Padang, where the overall toxR positive isolates have no tdh or trh gene (Zulkifli et al, 2009). However, some other studies seem to successfully detect the presence of these virulence genes in environmental samples (Sujeewa et al, 2009; Marlina et al, 2007).

CONCLUSION

All of 40 V. parahaemolyticus isolates isolated from beef samples marketed in Pasar raya Padang were having toxR gene, but none of them has tdh or trh gene.

BIBLIOGRAPHY

DePaola, A., C. A. Kaysner, J. Bowers and D. W. Cook, 2000, Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks inWashington, Texas, and New York (1997 and 1998),