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"Latar belakang: Malaria merupakan salah satu masalah kesehatan masyarakat, dapat menyebabkan kematian dan secara langsung menyebabkan anemia. Berdasarkan hasil Riskesdas 2013, insiden malaria di Indonesia1,9 . Upaya menekan angka kesakitan dan kematian dilakukan antara lain melalui penegakan diagnosis dini. Plasmodium falciparum dan Plasmodium vivax adalah 2 spesies penyebab utama penyakit malaria yang ditemukan di Indonesia. Malaria PF/PV Ag Cassette Test Star Diagnostic Plus dengan metode imunokromatografi mendeteksi antigen kedua spesies Plasmodium, sehingga dapat dipertimbangkan sebagai sarana diagnostik alternatif untuk mendiagnosis malaria. Penelitian ini bertujuan melakukan uji diagnostik Malaria PF/PV Ag Cassette Test Star Diagnostic Plus dan mencari korelasi hemolisis dengan derajat parasitemia.Metode: Desain penelitian adalah uji diagnostik menggunakan baku emas pemeriksaan mikroskopik pada 79 orang. Uji korelasi dilakukan pada 32 orang yang memenuhi kriteria inklusi dan eksklusi.Hasil: Pada penelitian ini, didapatkan nilai sensitivitas, spesifisitas, NPP, NPN Malaria PF/PV Ag Cassette Test Star Diagnostic Plus masing-masing sebagai berikut 69 /75 , 100 /100 , 100 /100 , dan 92 /97 . Uji korelasi tidak dapat dilakukan karena hanya 1 pasien yang mengalami hemolisis intravaskuler dan ekstravaskuler dengan derajat parasitemia sedang dan 2 pasien hemolisis ekstravaskuler dengan derajat parasitemia ringan.Kesimpulan:Malaria PF/PV Ag Cassette Test Star Diagnostic Plus dapat digunakan untuk membantu diagnosis malaria pada daerah yang tidak memiliki teknisi laboratorium yang trampil. Secara deskriptif terlihat bahwa hemolisis intravaskuler dan ekstravaskuler mulai terjadi pada derajat parasitemia sedang. Kata kunci: Malaria PF/PV Ag Cassette Test Star Diagnostic Plus ; hemolisis intravaskuler; hemolisis ekstravaskuler; derajat parasitemia.

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Background. Malaria is one of the public health problems that can cause death and directly cause anemia. Based on the results of Riskesdas 2013, the incidence of malaria in Indonesia is 1.9 . Attempts to reduce morbidity and mortality are among others through early diagnosis. Plasmodium falciparum and Plasmodium vivax are the two main causes of malarial disease found in Indonesia. Malaria PF PV Ag Cassette Test Star Diagnostic Plus with imunochromatography method detects both antigen of Plasmodium species so that it can be considered as an alternative diagnostic tool for diagnosing malaria. This study aims to perform diagnostic test Malaria PF PV Ag Cassette Test Star Diagnostic Plus and lookes for correlation between the degree of parasitemia and hemolysis.Methods. The study design was a diagnostic test using a gold standard microscopic examination in 79 people. Correlation test done on 32 people who meet the inclusion and exclusion criteria. Results. In this study, the values of the sensitivity, specificity, NPP, NPN Malaria PF PV Ag Cassette Test Star Diagnostic Plus were 69 75 , 100 100 , 100 100 , and 92 97 respectively. Correlation test can not be done because only one patient undergo intravascular and extravascular hemolysis with moderate degree of parasitemia and 2 patients have extravascular hemolysis with mild degree of parasitemia.Conclusion. Malaria PF PV Ag Cassette Test Star Diagnostic Plus can be used to support the diagnosis of malaria in areas that do not have a skilled laboratory technicians. Descriptively seen that intravascular and extravascular hemolysis begin to occur in the degree of moderate parasitemia. Keywords Malaria PF PV Ag Cassette Test Star Diagnostic Plus , intravascular hemolysis, extravascular hemolysis, degree of parasitemia."

"ABSTRAKHemolisis merupakan masalah yang umum dijumpai dalam praktik laboratorium dengan prevalensi 3,3 dari total spesimen yang diterima di laboratorium. Hemolisis memiliki pengaruh yang berbeda pada pemeriksaan PT dan APTT pada subyek sehat dan pasien Sysmex CS2100i merupakan alat koagulometer yang menggunakan prinsip deteksi koagulasi dengan transmisi cahaya foto-optikal yang dilengkapi dengan detektor hemolisis ikterik dan lipemik HIL dan multiple wavelength detector. Aspek terpenting dalam praktik laboratorium terkait hemolisis adalah mengetahui batasan indeks hemolisis yang dapat menimbulkan bias bermakna di dalam suatu pemeriksaan dalam hal ini PT dan APTT. Jumlah subyek penelitian sebesar 70 orang yang dibagi dua yaitu, kelompok sehat sebesar 35 orang dan kelompok sakit dengan warfarin sebesar 35 orang. Pembuatan hemolisat dilakukan dengan metode trauma mekanik menggunakan syringe insulin dengan jarum 30G. Pada hasil PT dan APTT subyek sehat didapatkan uji repeated measures ANOVA bermakna, p=0,001 dan subyek sakit dengan warfarin didapatkan uji Friedman bermakna, p=0,001. Uji post-hoc Dunnett subyek sehat untuk hasil PT didapatkan nilai bermakna pada konsentrasi hemolisis 150, 200, 250, 330 dan 500 mg/dL, sedangkan hasil APTT didapatkan hasil bermakna pada konsentrasi hemolisis 250, 330, dan 500 mg/dL. Uji post-hoc Wilcoxon subyek sakit untuk hasil PT didapatkan nilai bermakna pada konsentrasi hemolisis 100, 150, 200, 250, 330 dan 500 mg/dL, sedangkan hasil APTT didapatkan nilai bermakna pada konsentrasi hemolisis 250, 330 dan 500 mg/dL.Bias hemolisis maksimal yang masih dapat diterima dengan kriteria Ricos dkk untuk PT dan APTT subyek sehat masing-masing adalah 100 mg/dL, sedangkan subyek sakit dengan warfarin adalah 50 mg/dL dan 200 mg/dL. Batasan dengan kriteria CLIA untuk PT dan APTT subyek sehat adalah 330 mg/dL dan 250 mg/dL, sedangkan subyek sakit dengan warfarin adalah 330 mg/dL baik untuk PT maupun APTT. Dari grafik scatter didapatkan tren pemanjangan hasil PT dan APTT subyek sehat, sedangkan pada subyek sakit dengan warfarin didapatkan tren pemanjangan hasil PT dan pemendekan hasil APTT. Penerapan batasan bias hemolisis maksimal memungkinkan praktisi laboratorium untuk tetap menerima spesimen dengan interferensi hemolisis pada pemeriksaan PT dan APTT, memastikan hasil yang dikeluarkan tetap akurat, tanpa menunda penatalaksanaan terhadap pasien dan mengurangi biaya dan ketidaknyamanan yang timbul akibat pengambilan kembali spesimen.

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ABSTRACTHemolysis is a common problem in laboratory practice with a prevalence of 3.3 of the total specimens received in the laboratory. Haemolysis have a different influence on the examination of the PT and APTT in healthy and patients subjects. Sysmex CS2100i is a coagulometer with the photo optical method, equipped with hemolysis, icteric and lipemic detector HIL and multiple wavelength. The most important aspect in laboratory practice is to know the limits associated with haemolysis that can cause significant bias in PT and APTT assay. The total number of research subjects are 70 people, divided into 35 healthy subjects and 35 patient subject undergoing warfarin therapy. Hemolysate was conducted using a mechanical trauma using insulin syringe with 30G needle.. Repeated measures ANOVA test of PT and APTT on healthy subjects obtained a significant statistical result, p 0.001. The warfarin users also had a significant statistical result with Friedman test, p 0.001. Post hoc Dunnett test on PT values of healthy subjects, obtained a statisticaly significant results in hemolysis concentration of 150, 200, 250, 330 and 500 mg dL, while the APTT results obtained significant statistical results in haemolysis concentration of 250, 330, and 500 mg dL. Wilcoxon post hoc test of PT on patient subjects obtained significant result in the haemolysis concentration of 100, 150, 200, 250, 330 and 500 mg dL, while the APTT values obtained significant results in hemolysis concentration 250, 330 and 500 mg dL. The maximum bias that still could acceptable by Ricos et al criteria for PT and APTT on healthy subjects for both were 100 mg dL, whereas patient subjects undergoing warfarin therapy was 50 mg dL and 330 mg dL. Using CLIA criteria for PT and APTT on healthy subjects resulted maximum bias was 330 mg dL and 250 mg dL, whereas warfarin users was 330 mg dL for PT and APTT. There was a trend of increase in the readings of PT and APTT on healthy subjects, while on patient subjects undergoing warfarin there was a trend of increase in PT and decrease of APTT results. The application of acceptable hemolysis bias limit, enable laboratory practitioners to process hemolysis specimens in PT and APTT assays, ensuring the results is still accurate without delaying clinical decision and to reduce the cost and inconvenience arising from the specimen recollection. "

"Latar belakang: Kemampuan survival sel darah merah ini dapat dilihat dari hemolisis sel darah merah selama penyimpanan. Penurunan survival sel darah merah ini kemungkinan salah satunya karena masih adanya leukosit dalam komponen darah. Adanya leukosit dalam produk PRC dapat meningkatkan storage lesion sehingga menurunkan kemampuan survival sel darah merah ini. Selain mengurangi jumlah leukosit, upaya untuk menjaga kestabilan membran sel darah merah dilakukan dengan cara menggunakan manitol dalam additive solution.Metode : 40 sampel PRC yang terdiri dari kantong PRC, PRC+Filter, PRC+SAGM, PRC+SAGM+Filter yang disimpan dengan suhu 2°-6°C diperiksa kadar glukosa, pH dan hemolisis pada hari ke-0, 7, 14, 21, 28, 35, dan 42. Data hasil penelitian ini dianalisis menggunakan uji statistik ANOVA dengan batas kemaknaan <0,05.Hasil : Penggunaan filter untuk mengurangi jumlah leukosit didapatkan hasil yang bermakna (<0,05). Terjadi penurunan kadar glukosa, pH dan peningkatan hemolisis selama penyimpanan pada semua jenis kantong. Tingkat hemolisis produk darah PRC pada hari ke-35 telah lebih dari standar, sedangkan pada tiga produk PRC yang lain pada masa simpan 42 hari masih dalam batas normal.Kesimpulan : Baik pengurangan leukosit mau pun penambahan additive solution pada PRC dapat mempertahankan viabilitas sel darah merah sehingga pada penyimpanan hari ke-42.

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ABSTRACT

Background: The ability of red blood cell survival can be seen from hemolysis of red blood cells during storage. The decrease in survival of red blood cells is probably one of them because of the presence of leukocytes in the blood component. The presence of leukocytes in PRC products can increase storage lesion thereby reducing the survival of red blood cells. In addition to reducing the number of leukocytes, efforts to maintain the stability of the red blood cell membrane are carried out by using mannitol in additive solutions.

Methods: 40 PRC samples consisting of PRC bags, PRC+Filters, PRC+SAGM, PRC+SAGM+Filters stored at 2°-6°C were examined for glucose, pH and hemolysis levels on days 0, 7, 14 , 21, 28, 35, and 42. Data from the results of this study were analyzed using ANOVA statistical tests with significance limits <0.05.

Results: The use of filters to reduce the number of leukocytes showed significant results (<0.05). A decrease in glucose levels, pH and increased hemolysis during storage in all types of bags. The level of hemolysis of PRC blood products on the 35th day was more than standard, whereas in the other three PRC products the 42-day shelf life was still within normal limits.

Conclusion: Both leukocyte reduction and additive solution addition in PRC can maintain the viability of red blood cells so that at the 42nd day storage.

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"Akumulasi rantai globin-α berlebihan pada membran SDM thalassemia-β mayor menyebabkan hemolisis, eritropoiesis tidak efektif dan anemia kronik sehingga memerlukan transfusi sel darah merah (SDM) terus menerus. Transfusi rutin dan hemolisis mengakibatkan besi bebas sebagai radikal bebas dan membentuk radikal peroksil lipid di membran SDM sehingga memperberat hemolisis. Antioksidan α-tokoferol menghambat pembentukan radikal peroksil lipid tersebut.Penelitian ini bertujuan untuk menilai peran α-tokoferol terhadap hemolisis dan stres oksidatif. Penelitian ini merupakan uji klinis acak tersamar ganda pada thalassemia-β mayor usia 5–18 tahun yang mendapat transfusi dan kelasi besi rutin di Pusat Thalassemia RSUP dr.Ciptomangunkusumo Kiara. Intervensi plasebo dan α-tokoferol diberikan selama empat minggu. Suplementasi α-tokoferol berdasarkan rekomendasi Institute of Medicine (IOM), 4–8 tahun 200 mg/hari; 9–13 tahun 400 mg/hari; 14–18 tahun 600 mg/hari. Penilaian penanda hemolisis menggunakan haptogobin (Hp), hemopeksin (Hx) dan fragilitas osmotik SDM. Penanda stres oksidatif yaitu MDA, GSH, GSSG, rasio GSH/GSSG dan α-tokoferol. Pemeriksaan laboratorium dilakukan sebelum dan setelah diberikan plasebo/α-tokoferol, sesaat sebelum transfusi SDM. Analisis uji t-tidak berpasangan untuk melihat perbedaan antara kelompok studi dan uji korelasi untuk melihat hubungan antara variabel.Pada bulan Desember 2016 hingga Juli 2017, 40 subjek mampu menyelesaikan penelitian, 20 subjek kelompok plasebo dan 20 subjek kelompok α-tokoferol. Nilai rerata Hp lebih besar pada kelompok α-tokoferol (3,01 mg/dL) dibandingkan kelompok plasebo (1,08 mg/dL), secara statistik berbeda bermakna (p = 0,021). Nilai rerata kadar Hx dan persentase hemolisis SDM tidak berbeda bermakna pada kedua kelompok studi (p > 0,05). Tidak ada perbedaan bermakna pada kelompok α-tokoferol dan plasebo untuk kadar MDA (1,003 nmol/L dan 1,07 nmol/L), GSH (5,81 µM dan 6,15 µM), GSSG (1,77 µM dan 1,86 µM) dan rasio GSH/GSSG (1,29 dan 1,31), (P > 0,05).Antioksidan α-tokoferol dapat mengurangi hemolisis dan secara tidak langsung memperbaiki kadar Hp pada thalassemia-β mayor, akan tetapi tidak mampu memengaruhi stres oksidatif.

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The accumulation of excess unmatchedα-globin chains in the red blood cell membrane of β-thalassemia major leads to hemolysis, ineffective erythropoiesis and chronic anemia which needs multiple red blood cell transfusion. Routine transfusions may lead to iron overload as free radical in the red blood cell membrane, resulting clinically as severe hemolysis. Alpha-tocopherol as an antioxidant has been known as a potent scavenger of hydroxyl lipid radical.The aim of this study was to evaluate the effects of α-tocopherol in hemolysis and oxidative stress on the red cell membrane in β-thalassemia major. This randomized double-blind, placebo-controlled study was done in β-thalassemia major patients range aged 5–18 years old who regularly had transfusion and receiving iron chelating agents at Thalassemia centre, Kiara Ciptomangunkusumo Hospital. All subjects were randomized to receive either α-tocopherol or placebo orally for 4 weeks. Subjects in the experimental group received α-tocopherol, the doses based on the recommendation from Institute of Medicine (IOM) as follows: 200 mg/day for 4–8 years old; 400 mg/day for 9–13 years old; 600 mg/day for 14–18 years old. Laboratory analysis for hemolysis variables were haptoglobin, hemopexin, osmotic fragility test. Oxidative stress and antioxidant variables were MDA, GSH, GSSG, GSH/GSSG ratio, and α-tocopherol. All variable were evaluated before 4 weeks and after consuming α-tocopherol or placebo, just before they received a blood transfusion. The statistical analysis results using independent t-test and correlation test.During December 2016–July 2017, 40 subjects completed the study, they were 20 subjects in the placebo group and 20 subjects in the α-tocopherol group. There was significant enhancement of haptoglobin mean level in the α-tocopherol group (3.01 mg/dL) compared to placebo (1.08 mg/dL), (p = 0.021). The mean level of hemopexin and the percentage of RBC hemolysis did not significantly different in both groups, (p > 0.05). We also did not find any significantly different in mean level of MDA (1.003 nmol/L and 1.07 nmol/L), GSH (5.81 µM and 6.15 µM), GSSG (1.77 µM and 1.86 µM) and GSH/GSSG ratio (1.29 and 1.31), (p > 0.05) for the α-tocopherol and placebo groups.The effects of α-tocopherol may improve hemolysis and haptoglobin level indirectly in β-thalassemia major, but there was no significant role in oxidative stress."

"Tujuan umum: Mengetahui profil keamanan dan efek getah J. curcas terhadap jaringan gigi dan periapeks dalam persiapan untuk memanfaatkan pemakaian bahan alami getah J. curcas pada radang pulpa. Tujuan khusus (1) Mengetahui kandungan golongan senyawa getah J. curcas. (2) Mengetahui sitotoksisitas getah J. curcas. (3) Mengetahui toksisitas akut pemberian secara oral dosis tunggal getah J. curcas pada hewan percobaan. (4) Mengetahui aktivitas hemolisis getah J. curcas pada darah manusia secara in vitro. (5) Mengetahui sifat mutagenisitas getah J. curcas. (6) Mengetahui efek getah J. curcas terhadap pembebasan interleukin-1β oleh sel makrofag. (7) Mengetahui efek getah J. curcas terhadap pembebasan kolagenase pada set fibroblast. (8) Mengetahui efek histopatologik getah J. curcas terhadap pulpa dan jaringan periapeks gigi pada hewan percobaan. (9) Mengetahui efek getah J. curcas terhadap kekerasan macro jaringan keras gigi manusia secara in vitro. (10) Mengetahui efek getah J. curcas terhadap jaringan keras gigi manusia dalam hal kelarutan unsur kalsium dan fosfat secara in vitro. Metode penelitian: Disain penelitian eksperimental dan eksplorasi. Penelitian dibagi atas (1) skrining fitokimia, (2) tahap 1 dan (3) tahap 2 evaluasi biologik getah J. curcas. Untuk standardisasi getah J. curcas diambil dari satu petak tanaman dalam satu musim, kemudian diukur pH, volume basah, diliofilisasi, diukur berat kering, dan disimpan pada -20°C sebagai sampel. (1). Skrining fitokimia getah J. curcas. Analisis kualitatif golongan senyawa diidentifikasi dari ekstrak eter, etil asetat, dan air. (2). Uji toksisitas 1. Uji sitotoksisitas. (1) Metoga agar overlay. Getah J. curcas dan kontrol diserap oleh cakram selulosa, kemudian diletakkan di atas permukaan agar yang menutupi selapis sel Fib L929 yang telah diwarna neutral red. Evaluasi berdasar luas zona dekolorisasi dan zona lisis yang terbentuk setelah 24 jam. (2) Assay MTT. Getah J. curcas dalam medium diberikan pada kultur set Fib L929 cell line dan sel primer fibroblast gingiva manusia yang tumbuh dalam mikroplat 96-sumur. Setelah 1-4 hari, dilakukan assay MTT. Evaluasi berdasar perbandingan nilai OD kontrol dan perlakuan. 2. Uji toksisitas akut. Mencit diberi getah J. curcas secara intragastrik sebanyak 1 kali. Dihitung LD5O berdasar jumlah mencit yang mati. Dibandingkan antara kelompok perlakuan dan kontrol dalam hal tanda toksisitas, berat badan selama 2 minggu, pemeriksaan makroskopik dan mikroskopik organ tubuh. 3. Uji hemolisis. Darah dicampur dengan berbagai konsentrasi getah J. curcas. Evaluasi berdasar pembebasan hemoglobin, dibandingkan OD kelompok perlakuan dengan kontrol positif air, dan kontrol negatif salin. 4. Uji mutagenisitas. Getah J. curcas dikultur dengan bakteri S. typhi dan E. coil mutan. Evaluasi berdasar penghitungan koloni reversi bakteri, dibandingkan kelompok perlakuan, kontrol positif dan kontrol negatif. (3) Efek getah J. curcas terhadap makrofag dan fibroblast 1. Efek getah J. curcas terhadap pembebasan IL-1β. Lima dosis getah J. curcas dimasukkan ke dalam kultur makrofag peritoneum mencit BALB/c, secara bersamaan, sebelum, atau sesudah pemberian LPS. Setelah 1 dan 2 hari, IL-1β dalam supernatan diukur secara ELISA dengan Quantikine IL-1β for mouse kit. 2. Efek getah J. curcas terhadap pembebasan kolagenase oleh fibroblast. Empat dosis getah J. curcas dan IL-1β dimasukkan dalam kultur sel primer fibroblast gingiva manusia. Setelah 1-4 hari kolagenase dalam supematan diukur dengan assay kolagenase. Hasil degradasi kolagen dipisahkan dengan SDS-PAGE. Pita 3/4 αA diukur dengan program komputer Adobe Photo. (4) Efek histopatologik getah J. curcas pada jaringan pulpa dan periapeks. Getah J. curcas dimasukkan ke dalam kavitas gigi monyet. Setelah 3 hari, gigi diproses untuk pembuatan sediaan histologik. Evaluasi berdasar perbandingan pemeriksaan keadaan mikroskopik jaringan pulpa dan peripeks dalam hal inflamasi dan nekrosis, antara kelompok kontrol dan perlakuan. (5) Efek getah J. curcas terhadap jaringan keras gigi. 1. Efek getah J. curcas terhadap kekerasan mikro dentin dan email. Mahkota gigi premolar dibelah 4 longitudinal, lalu ditanam di dalam akrilik dengan 1 permukan tidak tertutup akrilik. Setelah direndam dalam 3 konsentrasi getah J. curcas, permukaan dentin dan email diberi indentasi oleh intan Knoop. Evaluasi berdasar perbandingan KHN kelompok kontrol dan perlakuan. 2. Efek getah J. curcas terhadap kelarutan kalsium dan fosfat. Mahkota gigi premolar utuh dibelah 4 secara longitudinal, lalu direndam dalam 3 konsentrasi getah J. curcas. Setelah 1-3 hari, kalsium dan fosfat yang larut dalam rendaman diukur berturut-turut dengan alat atomic absorption spectrophotometer (AAS) dan spektrofotometer (metoda asam askorbat). Hasil penelitian pH getah J. curcas rata-rata 3,49 ± 0,09 dan perbandingan berat kering/volume basah 15,12 ± 0,31%. (1) Skrining fitokimia: getah J. curcas mengandung golongan senyawa sterol, aglikon flavon, tanin, senyawa pereduksi, glikosida steroid, poliose, dan saponin. (2) Uji toksisitas 1.(1) Sitotoksisitas getah J. curcas pada metoda agar overlay ditemukan zona dekolorisasi indeks 2 dari 5 indeks zona. Tak ada lisis sel, bentuk sel masih jelas. (2) Assay MTT: pads getah J. curcas kadar 0,25% terhadap Fib L929 dan kadar 0,12% terhadap fibroblast gingiva, sel nekrosis. 2.(1) LD50 > 5 g/kg BB, sehingga getah J. curcas dapat diklasifikasi dalam toksik ringan. (2) Tidak ada perbedaan berat badan. (3) Tidak ada perbedaan makroskopik dan mikroskopik organ tubuh yang diperiksa. (4) Terjadi inaktivitas pada hari 1 pada kelompok perlakuan, selanjutnya tidak ada perbedaan. 3. Aktivitas hemolisis getah J. curcas 15% adalah 6,5% dibanding air. Tidak ada hemolisis pada konsentrasi getah J. curcas yang lebih rendah. 4. Tidak ada aktivitas mutagenisitas getah J. curcas. (3) Efek getah J. curcas terhadap makrofag dan fibroblast 1. (1) LPS meningkatkan pembebasan 1L-1β oleh makrofag. (2) Pemberian getah J. curcas menghambat pembebasan 1L-1β oleh makrofag. 2. (1) Makin lama waktu kultur, produksi kolagenase makin banyak. (2) Getah J. curcas menurunkan pembebasan kolagenase oleh fibroblast. (4) Efek histopatologik getah J. curcas terhadap jaringan pulpa dan periapeks (1) Inflamasi dan nekrosis terj adi pads daerah yang terbatas dekat dengan daerah yang kontak dengan getah J. curcas. Di bawahnya terdapat jaringan pulpa normal. (2) Tingkat inflamasi pulpa kelompok perlakuan tidak lebih parah dari kelompok kontrol. (3) Tidak ada radang periapeks pads kelompok kontrol dan perlakuan. (5) Efek getah J. curcas terhadap jaringan keras gigi. 1. Efek getah J. curcas terhadap kekerasan mikro dentin dan email. (1) Kekerasan mikro dentin tidak berbeda bermakna pada 1 dan 2 hari perendaman getah J. curcas antara kelompok kontrol dan perlakuan. Namur lebih kecil setelah 3 hari pada konsentrasi getah 15%. (2) Kekerasan mikro email tidak berbeda antara kelompok kontrol dan perlakuan pada 1 dan 3 hari, Namun lebih kecil setelah 2 hari pada konsentrasi getah J. curcas 15%. 2. Kadar kalsium dan fosfat yang larut meningkat sesuai dengan kenaikan konsentrasi getah J. curcas. Namun lama perendaman tidak berpengaruh secara bermakna terhadap kelarutan kalsium. Kesimpulan (1) Getah J. curcas mengandung sterol, aglikon flavon, tanin, senyawa pereduksi, glikosida steroid, poliose, dan saponin. (2) Tahap 1 evaluasi biologik: getah J. curcas relatif aman pada hewan percobaan berdasar LD50>5 g/kg BB sehingga termasuk dalam klasifkasi toksik ringan; hemolisis 6,5% dibanding air; tidak mutagen; dan sitotoksik dengan nekrosis koagulasi. (3) Uji tahap 2: getah J. curcas cukup efektif dalam menanggulangi pulpalgia, berdasar nekrosis pulpa terbatas, tidak ada kelainan periapeks; kekerasan mikro email dan dentin tidak turun pada 1 hari; menghambat pembebasan IL-1β dan kolagenase. Namun getah melarutkan kalsium dan fosfat. Kesimpulan penelitian: penelitian dapat dilanjutkan ke tahap uji klinik atau tahap 3.

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Biological Study on the Effects of Jatropha Curcas (Euphorbiaceae) Latex on Dental and Periapical TissuesObjective: The objective of this study was to evaluate the safety level and the effects of J. curcas latex on dental and periapical tissues. The aims in details were (1) to identify the main classes of chemical constituent in J. curcas latex; (2) to evaluate the cytotoxicity of J. curcas latex; (3) to determine the acute toxicity of J. curcas latex after single oral administration on mice; (4) to assess hemolytic activity of J. curcas latex; (5) to evaluate mutagenic activity of J. curcas latex; (6) to evaluate the effect on J. curcas latex of IL-1 il release from macrophages; (7) to evaluate the effect of J. curcas latex on collagenase release from fibroblasts; (8) to assess the histopathological effects of J. curcas latex on monkey dental pulp and periapical tissues; (9) to determine the effects of J. curcas latex to dentin and enamel micro-hardness; (10) to assess the effects of J. curcas latex on dissolving calcium and phosphate. Methods: Research design was experimental and explorative. To standardize the sample, J. curcas latex was collected from Balittro, Bogor in 1997, then the pH and wet volume were measured, the latex was lyophilized, dry weight was measured, and latex was stored at-20°C as sample. Biological evaluation was grouped into (1) phytochemical sreening, (2) toxicity test, (3) effects of J.curcas latex on cell, (4) effects of J.curcas latex on dental pulp and periapical tissues, and (5) effects of J.curcas latex on dental hard tissues, (1). Phytochemical screening: the main classes of chemical constituents of J. curcas latex were analyzed qualitatively from ether, ethyl acetate, and water extracts. (2). Toxicity test 1. Cytotoxicity test. (1) Agar overlay technique. J. curcas latex was imbibed in cellulose discs and put on the surface of agar overlaying a neutral red stained Fib L929 cell monolayer. Evaluation was judged on zone index and lysis index after 24 hours incubation. (2) MT assay. J. curcas latex was added to human gingival fibroblasts and Fib L929 cell culture in 96-well micro-plates. After 1-4 days of incubation, MTT assay was performed. Evaluation was based on comparing the OD values of control and test groups. 2. Acute toxicity. A single dose of J. curcas latex was given to male and female mice, intragastrically. LD50 was determined based on mortality rate. Assessment was also performed on 2 weeks observations of body weight, macroscopic and microscopic examinations of several organs. 3. Hemolysis test. Blood was mixed with several concentrations of J. curcas latex. The result was the extent of hemolysis expressed based on the absorbance of the test samples, negative and positive controls. 4. Mutagenicity test. L curcas latex was added to the S. ryphi and E. coil mutans culture. Assessment was based on bacterial revertant colonies, compare to positive and negative controls. (3) Effects of J.curcas latex on macrophages and fibroblasts 1. Effects of .T. curcas latex on the release of IL-1 β from macrophages. Five doses of J. curcas latex from 75-1200 μg/ml were added into the culture of BALB/c mice peritoneal macrophages, along with, after, or before addition of LPS. Following 1-3 days of incubation, IL-1P presence in supernatant was measured by ELISA using Quantikine ]L-1P for mouse kit. 2. Effects of J. curcas latex on the release of collagenase. Four doses of J. curcas latex from 37.5-300 µg/ml were added to human gingival fibroblasts cell culture. After 1-4 days of incubation, collagenase in the supernatant was assayed with collagen. The degradation products were then separated by SDS-PAGE and the density of 3/4 αA bands was measured semi quantitatively by Adobe Photo computer program. (4) Effects of J.curcas latex on dental pulp and periapical tissues. The latex of J. curcas was brought in contact with dental pulp and sealed. Assessment was based on the presence of inflammation and necrosis in dental pulp and periapical tissues, histopathologically. (5) Effects of J.curcas latex on dental hard tissues 1. Effects of J. curcas latex on dentin and enamel micro-hardness. Intact premolar crowns were cut longitudinally into 4 fragments, followed by embedding of each fragment in acrylats leaving 1 free surface. The fragments were then soaked in 3 concentrations of J. curcas latex from 3.7-15% for 1-3 days. The dentin and enamel micro-hardness were assessed by Knoop hardness measurement. 2. Effects of J. curcas latex on dissolved calcium and phosphate. Intact premolar crowns were cut longitudinally into 4 fragments, followed by soaking the fragments in 3 concentration of J. curcas latex from 3.7-15% for 1-3 days. The dissolved calcium and phosphate were measured according to atomic absorption spectrophotometer and spectrophotometer (ascorbic acid method), respectively. Results: The mean ± SD of J. curcas latex pH was 3.49 ± 0.09. The dry weight/wet volume was 15.12 ± 0.31%. (1). Phytochemical screening: sterols, flavone aglycones, tannins, reducing compounds, sterol glycosides, poliose, and saponins were identified in J. curcas latex. (2) Toxicity test 1. (1) Agar overlay technique. 2-5 mm decoloration zones were observed, indicating that J. curcas latex was cytotoxic. No lysis of cells was observed within the decolorized zone. (2) MTT assay. At 2.5 mg/ml J. curcas latex no living Fib L929 cells were observed, while the same result was shown at 1.2 mg/ml J. curcas latex on human gingival fibroblasts. 2. LD50 was more than 5 g/kg BW, hence dry J. curcas latex may be classified into mildly toxic substance. No significant body weight difference was observed. Macroscopic and microscopic examination on several organs showed no differences between test and control groups. 3. 6,5% hemolytic activity of 15% J. curcas latex compared to water was observed, while no hemolisis was observed with lower concentrations of latex. 4. No mutagenic ativity was observed with J. curcas latex. (3) Effects of J.curcas latex on macrophages and fibroblasts 1. (1) LPS increased the release of IL-1β. (2) J. curcas latex inhibited the release of IL-lβ from macrophages. 2. (1) The longer the duration of incubation, the more collagenase was released. (2) J. curcas latex decreased collagenase release by human gingival fibroblast. (4) Effects of I. curcas latex on dental pulp and periapical tissues. Inflammation and necrosis were observed in a limited area, which was in direct contat with J. curcas latex, underneath was normal pulp. Inflammation in the pulp of test group was not greater than in the control group. No inflammation or necrosis in periapical tissues was observed in all groups. (5) Effects of J. curcas latex on dental hard tissues 1. (1) The micro-hardness of dentin was not lowered after 1 and 2 days treatment, but lower after 3 days at 15% J. curcas latex. (2) The enamel microhardness was not decreased after 1 and 3 days immersion in J. curcas latex, but decreased after 2 days at 15% J. curcas latex. 2. The calcium and phosphate release were increased in accordance to the concentration of J. curcas latex. The duration of treatment did not influence the release of calcium, while it influenced the release of phosphate. Conclusions (1) J. curcas latex contains sterols, flavone aglycones, tannins, reducing compounds, sterol glycosides, poliose, and saponins. (2) Level 1 biological evaluation: J. curcas latex is relatively safe in animals based on LD50>5 g/kg BW, 6,5% hemolysis compared to water, not mutagenic, but cytotoxic with coagulative necrosis. (3) Level 2 biological evaluation: J. curcas latex seems to be effective in relieving pulpal pain. It caused coagulative necrosis in pulp, which was in direct contact with J. curcas latex while the tissue underneath was normal. It did not cause inflammation of periapical tissues, and did not lower the dentin and enamel micro-hardness after 1 day of exposure, but it lowered the microhardness after 3 days. It inhibited IL-1β and collagenase release. It dissolved dental calcium and phosphate."