Disertasi

Ekstrak Kaya Antosianin Bekatul Beras Hitam CempoIreng, Biomarker Molekuler, dan Aberrant Crypt Foci padaModel Mencit Kanker Kolorektal = Anthocyanin-Rich Extract of Cempo Ireng Black RiceBran, Molecular Biomarkers, and Aberrant Crypt Foci in aColorectal Cancer Mice Model.

Kanker kolorektal merupakan penyebab utama kematian akibat kanker di dunia.Senyawa bioaktif pangan, termasuk antosianin, telah banyak dikaji dalampenelitian praklinik, namun bukti in vivo yang mengaitkan perubahan biomarkermolekuler dengan lesi prakanker masih terbatas. Penelitian ini bertujuanmengevaluasi perbedaan ekspresi biomarker molekuler yang merepresentasikanjalur inflamasi (NF-κB), proliferasi (β-catenin), dan metabolisme lipid (ABCA1),serta pembentukan aberrant crypt foci (ACF), pada model mencit kankerkolorektal yang diberikan ekstrak kaya antosianin dari bekatul beras hitamvarietas Cempo Ireng. Studi ini merupakan penelitian praklinik eksploratif (proofof-concept). Penelitian eksperimental in vivo ini menggunakan total 20 mencitjantan BALB/c yang dibagi ke dalam empat kelompok (n = 5 per kelompok). Lesikolorektal diinduksi menggunakan azoxymethane (AOM) dosis tunggal 10mg/kgBB secara intraperitoneal dan dextran sodium sulfate (DSS) 2% dalam airminum selama tiga siklus. Ekstrak kaya antosianin bekatul beras hitam CempoIreng diberikan secara per oral melalui sonde intragastrik dengan animalequivalent dose (AED) sebesar 0,032; 0,096; dan 0,16 mg/gramBB. Induksikarsinogen dan pemberian ekstrak dilakukan secara bersamaan sejak hari ke-0 danberlangsung selama 10 minggu. Terminasi hewan percobaan dilakukan pada harike-71. Evaluasi meliputi pewarnaan metilen biru untuk identifikasi ACF,pemeriksaan histopatologi, serta analisis imunohistokimia ekspresi protein NF-κB, β-catenin, dan ABCA1. Data numerik disajikan sebagai nilai median danrentang persentil ke-25 hingga ke-75, mengingat ukuran sampel yang terbatas dandistribusi data yang tidak diasumsikan normal. Perbandingan ekspresi biomarkermolekuler dan jumlah ACF dilakukan dengan membandingkan masing-masingkelompok perlakuan terhadap kelompok kontrol negatif menggunakan ujinonparametrik Mann–Whitney U. Nilai p < 0,05 dianggap bermakna secarastatistik. Hasil penelitian menunjukkan bahwa ekspresi protein NF-κB padakelompok perlakuan memiliki nilai median sebesar 1,58% (0,02;10,31), 1,98%(0,02;9,17), dan 1,62% (0,02;6,97) pada dosis ekstrak 0,032; 0,096; dan 0,1 6mg/gramBB, dibandingkan dengan median 2,04% (0,07;8,89) pada kelompokkontrol negatif. Uji statistik menunjukkan bahwa perbedaan ekspresi NF-κB antarkelompok tidak bermakna secara statistik (p = 0,67), dengan rentang nilai yangrelatif lebar pada seluruh kelompok. Ekspresi protein β-catenin pada kelompokperlakuan menunjukkan median 0,19% (0,09;3,41), 0,65% (0,03;4,54), dan 0,42%(0,03;1,46), sedangkan kelompok kontrol negatif memiliki median 0,81%(0,10;2,94); perbedaan antar kelompok juga tidak bermakna secara statistik (p =0,40). Ekspresi protein ABCA1 pada kelompok perlakuan menunjukkan median0,55% (0,03;3,72), 0,50% (0,05;2,69), dan 0,58% (0,06;2,59), dibandingkandengan 0,70% (0,01;1,82) pada kelompok kontrol negatif, tanpa perbedaanbermakna secara statistik (p = 0,31) dan tanpa pola dosis–respon yang konsisten.Analisis jumlah ACF menunjukkan tidak terdapat perbedaan bermakna antarakelompok perlakuan dan kontrol negatif (p = 0,96). Secara keseluruhan, penelitianini menunjukkan bahwa pemberian ekstrak kaya antosianin dari bekatul berashitam varietas Cempo Ireng pada model mencit kanker kolorektal tidakmenghasilkan perbedaan bermakna secara statistik pada ekspresi biomarkermolekuler maupun jumlah ACF dalam durasi pengamatan penelitian ini. Temuanini menegaskan bahwa perubahan biomarker molekuler awal tidak selalu bersifatlinear atau simultan dengan perubahan lesi prakanker. Karena itu, diperlukan studilanjutan dengan ukuran sampel lebih memadai, durasi pengamatan lebih panjang,serta pendekatan analitik memungkinkan evaluasi hubungan temporal antaraperubahan biomarker molekuler dan perkembangan lesi prakanker.
Kata kunci: Antosianin; bekatul beras hitam; ACF; NF-κB; β-catenin; ABCA1


Colorectal cancer is a leading cause of cancer-related mortality worldwide.Bioactive food compounds, including anthocyanins, have been extensivelyinvestigated in preclinical studies; however, in vivo evidence linking molecularbiomarker alterations with preneoplastic lesions remains limited. This studyaimed to evaluate differences in the expression of molecular biomarkersrepresenting inflammatory (NF-κB), proliferative (β-catenin), and lipidmetabolism (ABCA1) pathways, as well as the formation of aberrant crypt foci(ACF), in a murine model of colorectal carcinogenesis treated with ananthocyanin-rich extract derived from rice bran of the black rice variety CempoIreng. This study was designed as an exploratory preclinical proof-of-conceptinvestigation. This in vivo experimental study employed a total of 20 male BALB/cmice, divided into four groups (n = 5 per group). Colorectal lesions were inducedusing a single intraperitoneal dose of azoxymethane (AOM; 10 mg/kg bodyweight), followed by administration of 2% dextran sodium sulfate (DSS) indrinking water for three cycles. The anthocyanin-rich black rice bran extract wasadministered orally via intragastric gavage at animal equivalent doses (AEDs) of0.032, 0.096, and 0.16 mg/g body weight. Carcinogen induction and extractadministration were initiated concurrently on day 0 and continued for 10 weeks.Animals were euthanized on day 71. Evaluations included methylene blue stainingfor ACF identification, histopathological examination, and immunohistochemicalanalysis of NF-κB, β-catenin, and ABCA1 protein expression in the proximal,medial, and distal colon. Numerical data are presented as median values withinterquartile ranges (25th–75th percentiles), given the limited sample size andnon-normal data distribution. Comparisons of molecular biomarker expressionand ACF counts were performed by comparing each treatment group with thenegative control group using the nonparametric Mann–Whitney U test. A p-value< 0.05 was considered statistically significant. The results demonstrated thatmedian NF-κB protein expression in the treatment groups was 1.58% (0.02–10.31), 1.98% (0.02–9.17), and 1.62% (0.02–6.97) for extract doses of 0.032,0.096, and 0.16 mg/g body weight, respectively, compared with 2.04% (0.07–8.89) in the negative control group. Statistical analysis indicated no significantdifferences in NF-κB expression among groups (p = 0.67), with wide variabilityobserved across all groups. Median β-catenin expression in the treatment groupswas 0.19% (0.09–3.41), 0.65% (0.03–4.54), and 0.42% (0.03–1.46), whereas thenegative control group exhibited a median of 0.81% (0.10–2.94); these differenceswere also not statistically significant (p = 0.40). Median ABCA1 expression in thetreatment groups was 0.55% (0.03–3.72), 0.50% (0.05–2.69), and 0.58% (0.06–2.59), compared with 0.70% (0.01–1.82) in the negative control group, showingno statistically significant differences (p = 0.31) and no consistent dose–responsepattern. Analysis of ACF counts revealed no significant differences between thetreatment and negative control groups (p = 0.96). Overall, this study indicatesthat administration of an anthocyanin-rich extract derived from Cempo Irengblack rice bran in a murine model of colorectal carcinogenesis did not result instatistically significant differences in molecular biomarker expression or ACFformation within the duration of observation. These findings underscore that earlymolecular biomarker alterations may not necessarily occur in a linear orsimultaneous manner with preneoplastic lesion development. Accordingly, furtherstudies with larger sample sizes, extended observation periods, and analyticalapproaches capable of evaluating the temporal relationships between molecularbiomarker changes and preneoplastic lesion progression are warranted.
Keywords: Anthocyanin; black rice bran; ACF; NF-κB; β-catenin; ABCA1