Pharmacogn J. 2020; 12(3): 452-458 A Multifaceted Journal in the field of Natural Products and Pharmacognosy Original Article www.phcogj.com In Vitro Anti-Amebic Activity of Cage Xanthones from Cratoxylum sumatranum Stem Bark Against Entamoeba histolytica Fendi Yoga Wardana1, Defi Kartika Sari2, Myrna Adianti2,3, Adita Ayu Permanasari2, Lidya Tumewu2, Tomoyoshi Nozaki4, Aty Widyawaruyanti2,5, Achmad Fuad Hafid2,5,* ABSTRACT Background: Amoebiasis is caused by Entamoeba histolytica, which is a pathogenic species living on human colon tissues. The development of new drugs for anti-amebic are still very needed for clinical treatment. Objective: This aims to identify the compounds in Cratoxylum Fendi Yoga Wardana1, Defi sumatranum for their anti-amoeba activity. Materials and Methods: In this study we used Kartika Sari2, Myrna Adianti2,3, bioactivity-guided isolation and structural analysis to identified anti-amebic compounds from Adita Ayu Permanasari2, Lidya dichloromethane extract of Cratoxylum sumatranum stem bark. Their anti-amebic activity was Tumewu2, Tomoyoshi Nozaki4, determined by an in vitro cell-based assay against Entamoeba histolytica and an enzymatic Aty Widyawaruyanti2,5, Achmad assay on NAD kinase. Results: Two known compounds from the cage xanthone groups, Fuad Hafid2,5,* namely cochinchinoxanthone (1) and cochinchinone D (2), were isolated. The structures of the cage xanthone compounds were established by extensive spectroscopic data analysis. 1Graduate Program of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Compound (1) showed the greatest level of anti-amebic activity both in cell-based and enzymatic Airlangga, Surabaya, 60115, INDONESIA. assay, yielding IC50 values of 4.57 and 12.17 µg/mL, respectively. In contrast, compound (2) 2Natural Product Medicine Research and yielded IC50 values of 5.19 and 12.60 µg/mL, respectively. Conclusion: When considering the Development, Institute of Tropical Disease, demonstrated anti-amebic activities, it becomes apparent that these compounds, isolated Universitas Airlangga, Surabaya, 60115, INDONESIA. from Cratoxylum sumatranum stem bark, have the potential to be further developed into 3Department of Health, Faculty of Vocational effective anti-amebic medicine against Entamoeba histolytica. Education, Universitas Airlangga, Surabaya, Key words: Amoebiasis, Bioactivity-guided isolation, Cratoxylum sumatranum, Entamoeba 60115, INDONESIA. histolytica, NAD kinase. 4Department of School of International Health, Laboratorium of Biomedical Chemistry, The University of Tokyo, Tokyo, B; pruniflorone M; 5′-demethoxycadensin G; 113-0033, JAPAN. INTRODUCTION 5Department of Pharmacognosy and 1,5-dihydroxy-8-methoxyxanthone; 1,5-dihydroxy- Phytochemistry, Faculty of Pharmacy, The genus Cratoxylum consists of tropical plant 6,7-dimethoxyxanthone; 1,3,6-trihydroxy-7- Universitas Airlangga, Surabaya, 60115, within the Hypericaceae family which is widespread methoxyxanthone; 1,3,5,6-tetrahydroxyxanthone; 1 INDONESIA. in tropical forests. Plant from the genus Cratoxylum 1,2,8-trihydroxyxanthone; 2,8-dihydroxy- been widely used for ethnomedicinal applications Correspondence 1-methoxyxanthone; cratoxyarborenone F; in South East Asia as a treatment of stomachache Achmad Fuad Hafid 1,7-dihydroxyxanthone; trapezifolixanthone; and diarrhea as well as for the prevention of gastric cochinchinoxanthone; 5-O-methylisojacareubin; Department of Pharmacognosy and 2,3 Phytochemistry, Faculty of Pharmacy, ulcers. Further work was able to isolate xanthone 2,4,6-trimethoxybenzophenone; 4-hydroxy-2,6- Nanizar Zaman Joenoes Building, compounds from C. cochinchinense, which was dimethoxybenzophenone; and annulatomarin.11 Universitas Airlangga, Surabaya 60115, reported to have bioactivity as an antioxidant, INDONESIA. anticancer agent and protein inhibitor of tyrosine Amoebiasis is a common infection in the human Phone no: +62-31-5933150; in diabetes.4-7 Preliminary research screening digestive tract. The diseaseis caused by E. histolytica, Fax: +62-31-5935249; 22 species of plants from Balikpapan Botanical which is a pathogenic species found on human colon E-mail: [email protected]; tissues. Amoebiasis is associated more strongly [email protected] Gardens, Kalimantan Indonesia, demonstrated with poor sanitation and lower socioeconomic History that dichloromethane extract from the stem bark of Cratoxylum sumatranum (Jack) Bl. has anti- status while climate plays a minor role, reflected in • Submission Date: 25-1-2020; amebic activity against E. histolytica cells with an its worldwide distribution. This poses major health • Review completed: 17-02-2020; IC value of 22.07 µg/mL, CC of 29.69 µg/mL challenges in China, Southeast Asia, and Western • Accepted Date: 27-02-2020. 50 50 and a Selective Index (SI) of 1.34.8 Phytochemical Latin America.12 In Indonesia, amoebiasis is found DOI : 10.5530/pj.2020.12.70 studies of C. sumatranum obtained xanthone in 10% of the population, resulting in 30 deaths 13 Article Available online and anthraquinino-benzophenone such as annually. Amoebiasis is often caused by the parasite http://www.phcogj.com/v12/i3 cratoxyarborenones A-F; cratoxyarborequinones E. histolytica, however only circa 10% to 20% of people infected with E. histolytica become symptomatic.14 To Copyright A-B; vismione B; 2,4,6-trihydroxybenzophenone 9 treat these infection, metronidazole is usually used © 2020 Phcogj.Com. This is an open- 4-O-geranyl ether; δ-tocotrienol; betulinic acid, 10 15 access article distributed under the terms and sumatranaxanthone A. Other compounds that for both intestinal and extraintestinal amoebiasis. of the Creative Commons Attribution 4.0 have also been isolated are cratosumatranone A-B; Although clinical resistance of metronidazole has International license. pruniflorone N; neriifolone B; isocudraniaxanthone not been conclusively demonstrated, resistance of B; 10-O-methylmacluraxanthone; E. histolytica to treatment with metronidazole in macluraxanthone; 5-O-methyl-2- case of amoebiasis has been reported.16 In addition, deprenylrheediaxanthone B; pancixanthone it has been shown by in vitro assay that this parasite Cite this article: Wardana FY, Sari DK, Adianti M, Permanasari AA, Tumewu L, Nozaki T, et al. In Vitro Anti-Amebic Activity of Cage Xanthones from Cratoxylum sumatranum Stem Bark Phcogj.com Against Entamoeba histolytica. Pharmacogn J. 2020;12(3):452-8. 452 Pharmacognosy Journal, Vol 12, Issue 3, May-June, 2020 Wardana, et al.: In Vitro Anti-Amebic Activity of Cage Xanthones from Cratoxylum sumatranum Stem Bark Against is easily adaptable to the metronidazole therapy.17 Therefore, there is a Anti-amebic cell based assay clear and urgent need for the development of new anti-amebic drugs for clinical treatment. The cells of HM-1:IMSS (clone 6) Entamoeba histolytica strain kindly provided by Prof. T. Nozaki (University of Tokyo) were cultivated The isolation and identification of active compounds from the stem in Bisate-Iron-Serum (BI-S) medium (Sigma) that was supplemented bark of C. sumatranum, as conducted in this study, was informed by the with 10% (v/v) bovine serum (Sigma) and 1% (v/v) Diamond Vitamin- concept of bioactivity-guided isolation regarding anti-amebic activity.18 Tweena solution (JRH Biosciences, USA) at 37 °C. The cells were The anti-amebic testing was conducted in vitro according to a cell- conditioned for 2 days to reach a confluence of 80%. The Entamoeba based assay on inhibition of E. histolytica cell growth and an enzymatic histolytica cells were seeded in 98-well plates. 200 μL of cells and BI-S assay examining inhibition of NAD kinase activity, which has a role in medium were added into each well, then the wells were incubated 2 the metabolic processes of E. histolytica.19 hours at 35.5 °C. After 2 hours of incubation, they were replaced with mixtures of medium and sample (2.5 μL of extract or isolate), then MATERIALS AND METHODS incubated for 24 hours. The medium was replaced with 10% WST-1 General experimental procedures reagent (Roche, Germany) in warmed OPTI-MEM medium (Gibco- Life Technologies). After wards, they were incubated for 30 minutes at 1D and 2D NMR spectra were recorded on a JEOL (400 MHz), 37 °C and absorbance was measured at 560 nm using a GloMax reader. using CDCl3 as the solvent and tetramethylsilane (TMS) as the internal The percent inhibition of cells growth in each samples was calculated by standard. The LC system coupled with Q-TOF/MS (Agilent) was comparison to the control, the Probit analysis with SPSS statistical was used for HRESIMS analysis. The HPLC (Shimadzu, LC-06) included used to determine the 50% inhibitory concentration (IC50). a detector PDA SPD-M20A, pump LC-6AD and column using silica Anti-amebic enzymatic assay gel RP-18 shim pack 4.6 x 250 mm (Merck). Optical rotations were measured on a P1000-LED polarimeter. Column chromatography (CC) The Master Mix solution was prepared (a mixtures of ATP, INT was carried out on silica gel 60 (Merck). Pre-coated plates of silica gel media, tris HCl, miliQ water and NAD kinase/NO1 enzyme). 96 μL/well 60 GF254 were used for TLC analysis. Bioactivity assays were carried of the Master MIX solution was added to 96 well-plates. Subsequently, out on a GloMax Microplate Multidetection Reader (Promega). All each well was amended with 5 μL of sample (extract or isolate), 14 chemicals were analytical grade. μL of NADH solution and tris HCl with pH 7.5. The plates were then incubated for 10 minutes at room temperature, and absorbance was Plant material