2.

4 Anti-Helicobacter pylori assay

Helicobacter pylory ATCC 43504 and five clinical isolates from Panamanian dyspeptic outpatiens
were grown in 20.0 ml of Brucella (Difco, Co.)broth supplemented with 10% fetal calf serum and
yeast extract for 48 in a 10% CO2 atmosphere. The cells were gently centrifuged, washed twice with
PBS and concentrated to #3 Mac Farland equivalent. Bacillar morphol-ogy was assessed with Gram
staining. Neither coccoid forms nor contamination was detected (Lee and Megraud, 1996. An
adaptation of a 24 micro-well format using Tetrazolium egg-yolk agar was introduced (Vasquez et al,
1996). Briefly, 3.0 mg of the compound to be tested were dissolved in 1.0 ml of DMSO and finally to a
volume of 10 ml with the media. Serial double fold dilutions were done triplicate and seeded with
10l of the bacterial suspension. MICs were assessed by the absence of growth wvidenced by the
absence of salt reduction (Eloff, 1998; Rivas, 2000; Rivas at al, 2003). Amoxicilin was used as a
refrence compound.

2.5. Antiparasitic assays

The purecompound 1 was also screened in the antimalarial, anti-leishmanial and anti-trypanosomal
screens

2.5.1 Antimalarial activity

Antiplasmodial activity was determined in a chloroquine-resistant Plasmodium falciparum strain (w2

indochina) utiliz-ing a novel microfluorimetric assay to measure the inhibition of the parasite growth
based on the detection of the parasitic DNA by intercalation with PicoGreen (Corbett et al, 2004).
The IC 50 values were calculated from relative fluorescence units as compared with untreated
controls. The parasites were main-tained at 2% haematocrit in flat-bottomed flasks (75ml) with RPMI
1640 medium (Gibco BRL) supplemented with 10% human serum. Chloroquine was used as a
standard antimalarial agent.

2.5.2 Anti-leishmanial activity

The study used a colorimetric Leishmania mexicana (WHO-MOHM/B2/82/BELZ) promastigote growth

assay which is based upon the reduction of tetrazolium

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2.4 Anti-Helicobacter pylori assay

Helicobacter pylory ATCC 43504 dan lima isolat klinis dari outpatiens dispepsia Panama ditumbuhkan
dalam 20,0 ml Brucella (Difco, Co) kaldu ditambah dengan 10% serum janin anak sapi dan ragi ekstrak
selama 48 dalam suasana CO2 10%. Sel-sel lembut disentrifugasi, dicuci dua kali dengan PBS dan
terkonsentrasi ke # 3 Mac Farland setara. Bacillar morphol-ogy dinilai dengan pewarnaan gram. Baik
bentuk coccoid atau kontaminasi terdeteksi (Lee dan Megraud, 1996. Sebuah adaptasi dari format 24
mikro-baik menggunakan Tetrazolium kuning telur agar diperkenalkan (Vasquez et al, 1996). Secara
singkat, 3,0 mg senyawa yang akan diuji dilarutkan dalam 1,0 ml DMSO dan akhirnya volume 10 ml
dengan media. Serial pengenceran lipat ganda dilakukan rangkap tiga dan unggulan dengan 10 l
suspensi bakteri. MIC dinilai oleh adanya pertumbuhan wvidenced oleh adanya garam reduksi (Eloff,
1998; Rivas, 2000; Rivas di al, 2003). Amoxicilin digunakan sebagai senyawa refrence.
2.5. Tes antiparasit

Layar yang purecompound 1 juga diputar di antimalaria, anti-leishmanial dan anti-trypanosomal

2.5.1 Kegiatan antimalaria

Aktivitas antiplasmodial ditentukan dalam Plasmodium falciparum galur chloroquine-resistant (w2

indochina) utiliz-ing alat tes microfluorimetric baru untuk mengukur penghambatan pertumbuhan
parasit berdasarkan deteksi DNA parasit dengan interkalasi dengan PicoGreen (Corbett et al, 2004) .
The nilai IC50 dihitung dari unit fluoresensi relatif dibandingkan dengan kontrol yang tidak diobati.
Parasit yang main-tained sebesar 2% hematokrit dalam termos datar dipercaya (75ml) dengan
medium RPMI 1640 (Gibco BRL) ditambah dengan 10% serum manusia. Klorokuin digunakan sebagai
agen antimalaria standar.

Kegiatan 2.5.2 Anti-leishmanial

Penelitian ini menggunakan Leishmania mexicana kolorimetri (WHO-MOHM / B2 / 82 / BELZ) assay

pertumbuhan promastigote yang didasarkan pada pengurangan tetrazolium