8:47 AM | Author: Alicia Komputer

METODE PENELITIAN

  1. Tempat dan waktu penelitian

1. Tempat penelitian

Laboratorium Farmasi UIN Syarif Hidayatullah, Jakarta

2. Waktu penelitian

Penelitian ini berlangsung ± 2 bulan

  1. Alat penelitian

Alat yang digunakan adalah corong Buncher, neraca analitik, blender, penguap putar (vacum evaporator), kapas steril, oven (pemanas), botol timbangan, krus platina, eksikator, mortir, alu, timbangan, kandang hewan uji, alat bedah steril (gunting, pisau bedah, pinset, papan bedah dll), alat suntik steril, cawan petri, jarum ose, mikropipet, vial, kaca objek, pH meter/indikator pH, kotak preparat, pipet eppendorf, tip pipet eppendorf, laminar flow, hemositometer, sentrifuge, mikroskop cahaya, spektrofotometer, inkubator serta peralatan gelas steril yang lazim digunakan dilaboratorium.

  1. Bahan penelitian

Bahan yang digunakan adalah daun sirih yang diperoleh dari Balai Penelitian Tanaman Rempah Obat (BALITRO), gambir, HCL encer, Air-Kloroform, etanol 70%, larutan methanol dan air suling.

Bahan yang diperlukan untuk uji imunomodulator : makrofag yang berasal dari peritoneum mencit putih (Mus Musculus), bakteri uji Staphylococcus epidermis, Nutrien Broth (NB) DIFCO, eter, NaOH 0,1 N, HCL 0,1 N, metanol, pewarna giemsa, larutan phosphate buffered saline (PBS), larutan EDTA 0,2 M, dimetil sulfoksida (DMSO), imboost sirup (soho) 250 mg/5 ml, minyak imersi.


  1. Metode Penelitian

1. Determinasi tanaman

Bahan yang digunakan adalah daun sirih dan gambir yang di peroleh dari Balai Penelitian Tanaman Rempah Obat. Sebelum dilakukan penelitian terhadap tumbuhan, terlebih dahulu dideterminasi untuk mengidentifikasikan jenis dan memastikan kebenaran simplisia. Determinasi dilakukan di Herbarium Bogoriense, Balitbang Botani-Puslitbang Biologi LIPI Bogor.

2. Penyiapan bahan yang digunakan

a. Pengumpulan dan penyediaan simplisia

Simplisia kering yang diperoleh dari Balai Penelitian Tanaman Rempah Obat, kemudian simplisia kering diblender sampai menjadi serbuk, serbuk yang diperoleh disimpan dalam wadah bersih dan tertutup rapat.(2)

b. penyiapan bahan pereaksi

1) larutan HCl encer 10%

226 ml HCl P ditambahkan dengan air suling sampai volume 1000 ml.(3)

2) larutan air kloroform

Kloroform P 2,5 ml ditambahkan dengan air suling sampai volume 1000 ml dan dikocok hingga homogen.(3)

3) larutan EDTA 0,2 M

73,41 gr EDTA ferric monosodium salt dilarutkan dalam air suling sampai volume 1000 ml

4) larutan phosphate buffered saline (PBS) dengan pH ± 7,8

NaCl 8 gr, KCl 0,2 gr, Na2HPO4 1,15 gr, KH2PO4 0,2 gr dilarutkan dalam air suling sampai volume 1000 ml , atur pH ± 7,8

5) larutan giemsa 10%

10 gr Giemsa dilarutkan dalam 1000 ml air suling

6) larutan biru tripan

NaCl 0,81 gr + Na2HPO4 0,06 gr dilarutkan dalam 100 ml air suling. 0,4 gr biru tripan dilarutkan dalam 100 ml campuran NaCl dan Na2HPO4

3. Pemeriksaan mutu simplisia

1) Susut pengeringan

Simplisia ditimbang secara seksama 2 gr dan dimasukkan kedalam botol timbang dangkal bertutup yang sebelumnya dipanaskan pada suhu 1050 C selama 30 menit dan telah ditara sebelum ditimbang dengan menggoyang-goyangkan botol hingga merupakan lapisan setebal kurang lebih 5 mm sampai 10 mm, kemudian dimasukkan kedalam lemari pengering (oven) pengeringan dilakukan sampai bobot tetap, ditimbang.

2) Kadar abu

Ditimbang dan digerus sebanyak 2 gr zat, dimasukkan kedalam krus platina atau krus silikat yang telah dipijarkan perlahan-lahan hingga arang habis, dinginkan, ditimbang. Jika arang tidak dapat hilang, tambahkan air panas, saring dengan menggunakan kertas saring bebas abu. Pijarkan sisa abu dan kertas saring dalam krus yang sama, masukan filtrat didalam krus, uapkan, pijarkan hingga bobot tetap, ditimbang. Dihitung kadar abu terhadap bahan yang telah dikeringkan diudara.

3) Kadar abu tidak larut asam

Abu yang diperoleh pada penetapan kadar abu, didihkan dengan 25 ml HCl encer selama 5 menit, dikumpulkan bagian yang tidak larut dalam asam, disaring melalui krus kaca masir atau kertas saring bebas abu, cuci dengan air panas, dipijarkan hingga bobot tetap, ditimbang. Dihitung kadar abu yang tidak larut dalam asam terhadap bahan uang telah dikeringkan diudara.


4) Kadar sari larut air

Keringkan serbuk simplisia diudara, ditimbang 2 gr serbuk simplisia dimaserasi selama 24 jam dengan 100 ml air-kloroform, menggunakan labu bersumbat sambil dikocok selama 6 jam pertama dan kemudian dibiarkan selama 18 jam. Disaring, diuapkan filtrat hingga kering dalam cawan dangkal berdasar data yang telah ditara, dipanaskan sisa pada suhu 1050 C hingga bobot tetap. Dihitung kadar dalam persen sari yang larut dalam air, dihitung terhadap bahan yang telah dikeringkan di udara.

5) Kadar sari larut etanol

Keringkan serbuk simplisia di udara, ditimbang 2 gr serbuk simplisia dan dimaserasi selama 24 jam dengan 100 ml etanol 95%, menggunakan labu bersumbat sambil berkali-kali dikocok selama 6 jampertama kemudian dibiarkan selama 18 jam. Disaring cepat dengan menghindarkan penguapan etanol 95%, diuapkan 25 ml filtrat dalam cawan kering berdangkal rata yang telah ditara, dipanaskan sisa pada suhu 1050 C hingga bobot tetap. Dihitung kadar dalam persen sari yang larut dalam etanol 95%, dihitung terhadap bahan yang telah di keringkan di udara. (4)

4. Pembuatan ekstrak

a. Ekstrak etanol 70%

Serbuk halus simplisia sebanyak 100 gr dimaserasi dengan pelarut etanol 70% sambil diaduk kemudian disaring dengan menggunakan corong buncher. Maserasi dilakukan sampai tidak ada lagi senyawa-senyawa yang tersari dalam etanol 70% atau sampai filtratnya jernih. Filtrat yang diperoleh kemudian diuapkan dengan penguap putar (vacum evaporator) pada suhu 600 C, sampai pelarut tidak tersisa lagi dan didapat ekstrak kental. Ekstrak kental dikeringkan dengan oven pada suhu 700 C sampai kering. Dihitung nilai hasil rendemen ekstrak.


b. Ekstrak air

Serbuk halus simplisia sebanyak 100 gr dimasukan kedalam aquadest, dipanaskan selama 15 menit pada suhu 960 C, disaring. Ampas yang diperoleh dibilas hingga airsaringan tidak berwarna, ekstrak air yang didapat dipekatkan dengan menggunakan cawan porselen yang diletakan diatas tangas air dan dipanaskan pada suhu 700 C hingga pelarut tidak tersisa lagi dan didapatkan ekstrak kental. Ekstrak kental yang didapat dikeringkan dalam oven pada suhu 700 C. dihitung nilai hasil rendemen ekstrak. (2,5)

5. Uji imunomodulator

a. Aklitimasi hewan coba

Sebelum hewan coba digunakan terlebih dahulu diadaptasikan terhadap lingkungan dan dilakukan penimbangan berat badan setiap harinya diamati kondisi kesehatannya. Hewan coba diaklitimasi selama ± 14 hari, hewan coba hanya diberi makan dan minum saja.

b. Penyiapan ekstrak uji

1) Disiapkan vial kosong yang akan digunakan untuk pengujian, tiap ekstrak dilakukan pengujian pada konsentrasi 1000, 100, 10, 1, 0,1 ppm dan tiap konsentrasi dilakuk Dibuat larutan induk kedua dengan konsentrasi 100 ppm pipet 0,5 ml dari larutan induk pertama (1000 ppm) dan larutkan dalam 5 ml aquadest steril.

2) SEkstrak ditimbang ± 10 mg dan dilarutkan dalam 10 ml aquadest steril. Untuk membuat larutan induk dengan konsentrasi 1000 ppm. Untuk mempermudah kelarutan ekstrak uji dalam air sebelumnya ditambahkan dimetih sulfoksida (DMSO) 1-5 tetes.

3) Dibuat larutan induk kedua dengan konsentrasi 100 ppm pipet 0,5 ml dari larutan induk pertama (1000 ppm) dan larutkan dalam 5 ml aquadest steril.

4) Dibuat larutan induk ketiga dengan konsentrasi 10 ppm pipet 0,5 ml dari larutan induk kedua (100 ppm) dan larutkan dalam 5 ml aquadest steril.

5) Dibuat larutan induk keempat dengan konsentrasi 1 ppm pipet 0,5 ml dari larutan induk ketiga (10 ppm) dan larutkan dalam 5 ml aquadest steril.

6) Dibuat larutan induk kelima dengan konsentrasi 0,1 ppm pipet 0,5 ml dari larutan induk keempat (1 ppm) dan larutkan dalam 5 ml aquadest steril.

c. Penyiapan suspensi bakteri

1) sterilisasi peralatan dan bahan

alat-alat yang akan digunakan pada proses uji imunomodulator dan pembuatan media termasuk cawan petri, tabung reaksi kosong, gelas piala, tip pipet mikro, pipet volumetrik, batang pengaduk kaca disterilkan dalam oven pada suhu 1600 C selama ± 2 jam. Bahan yang digunakan seperti phosphate buffered saline (PBS) pH = 7, 8, air suling, medium disterilkan dalam autoklaf pada suhu 1210 C selama 15 menit, sedangkan ruangan yang akan digunakan dipastikan dalam keadaan steril, nyalakan lampu UV pada laminar flow ± 2 jam sebelum digunakan.

2) persiapan medium

medium pertumbuhan bakteri uji Staphylococcus epidermis adalah Mueller Hinton Medium (MHM) dengan komposisi sari pati sapi 300 gr, teknik 17,5 gr, starch 1,5 gr, bacto agar 17 gr, suspenskan 38 gr dalam 1 liter air suling pH akhir ± 7,3. Media Nutrient Broth (NB) = pH ± 6,8. suspensikan 8 gram dalam 1 liter air suling. Medium-medium tersebut kemudian diatur pHnya, lalu disterilkan dalam autoklaf 1210 C selama 15 menit.

3) penyiapan suspensi bakteri uji Staphylococcus epidermis

setelah 24 jam masa inkubasi, bakteri uji Staphylococcus epidermis telah tumbuh dan berkembang biak, diambil koloni yang telah tumbuh pada agar miring medium MHM dengan menggunakan ose steril. Ditanam koloni bakteri uji Staphylococcus epidermis pada medim NB cair secara aseptik, inkubasikan pada suhu 370 C selama 24 jam, kemudian disentrifugasi selama 1 jam dengan kecepatan 50.000 rpm. Cairan supernatant yang terbentuk pada bagian atas tabung reaksi di buang. Endapan bakter yang diperoleh disuspensikan dalam 10 ml phosphate buffered saline (PBS) steril pH = 7,8 kemudian dilakukan penentuan jumlah bakteri yang diperoleh secara spektrofotometrik (λ = 620 nm. Transmitan 10%) dan didapat jumlah bakteri setara dengan 109 sel/ml.

d. Dengambilan makrofag dari peritoneum mencit(6,7)

Mencit dieuthanasia dengan menggunakan eter (dimasukan kedalam toples yang berisi kapas yang telah dibasahi eter), diletakan pada papan bedah, dibersihkan bagian perut dengan etanol 70% dan bedah bagian perut dengan menggunakan alat bedah steril, jika ditemukan cairan peritoneum dalam jumlah sedikit pada perut ditambahkan larutan phosphate buffered saline (PBS) steril sebanyak 1-2 ml, aduk dengan hati-hati kemudian diambil cairan peritoneum sel makrofag dengan bantuan mikropipet. Disetarakan jumlah sel makrofag dengan alat hemositometer, hingga didapatkan populasi makrofag 107 sel makrofag /ml.

e. Uji viabilitas

Uji viabilitas makrofag dihitung dengan menggunakan hemositometer. Dihitung jumlah sel makrofag yang hidup (terwarnai) dan jumlah sel makrofag yang mati (terwarnai) dalam daerah 1-4. pewarnaan menggunakan larutan biru tripan. Persen viabilitas dapat dihitung dengan menggunakan (1)

f. Pengujian fagositosis dan pewarnaan giemsa (8,9)

1) Suspensi bakteri uji Staphylococcus epidermis 109 sel/ml 200 µl, makrofag peritoneum mencit 107 sel/ml sebanyak 200 µl dan ekstrak uji 200 µl dengan konsentrasi yang berbeda yaitu 0,1, 1, 100, 1000 ppm. Inkubasikan selama 30 menit dalam suhu 370 C, ditambahkan 50 µl larutan EDTA 0,2 M.

2) Sebagai kontrol positif digunakan suspensi bakteri uji Staphylococcus epidermis 109 sel/ml 200 µl, makrofag peritoneum mencit, 107 sel/ml 200 µl dan obat imunomodulator sebanyak 200 µl. sdangkan control negatif digunakan suspensi bakteri uji staphylococcus epidermis 109 sel/ml sebanyak 200 µl, makrofag peritoneum mencit 107 sel/ml sebanyak 200 µl tanpa pemberian ekstrak uji.

3) Buat preparat ulas kemudian fikasasi dengan metanol, biarkan selama 5 menit dan dibuang kelebihan metanol.

4) Warnai dengan mencelupkannya kedaalm larutan pewarna Giemsa 10% Amati hasil dibawah mikroskop cahaya. Dihitung aktivitas dan kapasitas fagositosis makrofag.

g. Penetapan nilai aktivitas dan kapasitas fagositosis (6)

1) Nilai aktivitas fagositosis menurut jumlah sel PMN dan sel makrofag yang secara aktif melakukan proses fagositosis dalam 100 sel dengan banyaknya sel makrofag yang dinyatakan dalam persen.

2) Nilai kapasitas fagositosis

Nilai kapasitas fagositosis adalah jumlah bakteri yang difagosit oleh 50 makrofag.

h. Uji efek sinergis

1) uji potensiasi ektrak daun sirih (Piper betle)

2) uji potensiasi ekstrak gambir (Uncaria gambir)

3) uji efek sinergis kombinasi antara ekstrak daun sirih (Piper betle) dan gambir (Uncaria gambir)


E. Teknik Analisa Data

Hasil uji fagositosis dan pewarnaan giemsa dihitung nilai aktivitas dan kapasitas fagositisis makrofag, yaitu :

1. Nilai aktivitas fagositosis

Nilai aktivitas fagositosis menurut jumlah sel makrofag yang secara aktif melakukan proses fagositosis dalam 100 sel dengan banyaknya sel makrofag yang dinyatakan dalam persen.

2. Nilai kapasitas fagositosis

Nilai kapasitas fagositosi adalah jumlah bakteri yang difagosit oleh 50 makrofag.

Data-data yang diperoleh dianalisa dengan menggunakan program pengolahan data statistik SPSS 12. pada analisa data ini ditentukan terlebih dahulu homogennitas sample dan normalitas data dari setiap variabel dan dilanjutkan dengan uji parametik anova satu arah dengan taraf signifikansi 95% apabila terdapat perbedaan yang bermakna maka dilanjutkan dengan uji membandingkan antar kelompok.(3)

Hipotesis :

H0 : tidak ada perbedaan yang bermakna antara tiap kelompok perlakuan

Ha : terdapat perbedaan yang bermakna antara tiap kelompok perlakuan

Criteria pengujian :

Bila nilai sig <>

Bila nilai sig > 0,05 H0 diterima, berarti tidak terdapat perbedaan

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