Pharmacogn J. 2017; 9(1): 8-13 Original Article A Multifaceted Journal in the field of Natural Products and Pharmacognosy www.phcogj.com | www.journalonweb.com/pj | www.phcog.net Cytotoxic Compounds from Kibatalia gitingensis (Elm.) Woodson Mariquit M. De Los Reyes1,2, Glenn G. Oyong3, Vincent Antonio S. Ng4, Chien-Chang Shen5, Consolacion Y. Ragasa4,6 Mariquit M. De Los Reyes1,2, Glenn G. Oyong3, Vincent Antonio S. Ng4, Chien-Chang Shen5, Consolacion Y. Ragasa4,6 1 Biology Department, De La Salle University Science & Technology Complex, Leandro V. Locsin Campus, Biñan City, Laguna 4024, PHILIPPINES. 2 Biology Department, De La Salle University, 2401 Taft Avenue, Manila 0922, PHILIPPINES. 3 Center for Natural Science and Environmental Research, De La Salle University, 2401 Taft Avenue, Manila 0922, PHILIPPINES. 4 Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 0922, PHILIPPINES. 5 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, 155-1, Li-Nong St., Sec. 2, Taipei 112, TAIWAN. 6 Chemistry Department, De La Salle University Science & Technology Complex, Leandro V. Locsin Campus, Biñan City, Laguna 4024, PHILIPPINES. Correspondence Mariquit M. De Los Reyes, Ph.D, Biology Department, De La Salle University Science & Technology Complex, Leandro V. Locsin Campus, Biñan City, Laguna 4024, PHILIPPINES. Telephone: +63 49 554 8900 Local 112; +632 536-0228 E-mail: mariquit.delosreyes@dlsu.edu.ph History • Submission Date: 08-07-2016; • Review completed: 22-07-2016; • Accepted Date: 10-08-2016. DOI : 10.5530/pj.2017.1.2 Article Available online http://www.phcogj.com/v9/i1 Copyright © 2016 Phcog.Net. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. ABSTRACT Ursolic acid (1), squalene (2), a mixture of α-amyrin acetate (3a) and lupeol acetate (3b), and isoscopoletin (4), isolated from the dichloromethane extracts of the leaves and twigs of Kibatalia gitingensis, were evaluated for their cytotoxic activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal fibroblast-neonatal (HDFn), using the in vitro PrestoBlue® cell viability assay. Compounds 1-4 exhibited strong cytotoxic activities against HT-29 cells with IC50 values ranging from 0.6931 to 1.083 μg/mL. Furthermore, 1-4 were moderately cytotoxic against HCT-116 cells with IC50 values ranging from 4.065 to 11.09 μg/mL. These compounds were least cytotoxic against MCF-7 cells with IC50 values ranging from 8.642 to 25.87 μg/mL. The most cytotoxic against HT-29 cells, HCT-116 cells and MCF-7 cells are 2, 4 and 1, respectively. Key words: Kibatalia gitingensis, Apocynaceae, Ursolic acid, Squalene, α-amyrin acetate, Lupeol acetate, Isoscopoletin, Cytotoxicity, MCF-7, HCT-116, HT-29, HDFn, PrestoBlue® cell viability assay. INTRODUCTION Kibatalia gitingensis (Elm.) Woodson, of the family Apocynaceae, is native to the Philippines where it is locally known as “laniti” or “laneteng-gubat”. It is classiied as vulnerable in the IUCN Red List of hreatened Species.1 It is commonly used to make building materials and decorative carvings and is also known to contain medicinal properties due to its alkaloid content.2 he leaves of K. gitingensis yielded a steroidal alkaloid, gitingensine, which was found to exhibit antispasmodic activity3,4 and ataraxic properties, able to tranquilize smooth muscles as well as vasodilate arteries of the skeletal muscles and the splanchnic region.5 Other studies reported that the leaves of K. gitingensis contain kibataline6,7 and 20-(epi-N-methyl) paravallarine.8 he plant contains an azasteroidal alkaloid which caused spontaneous motility in mouse and dog intestines and likewise removed serotonin-induced contractions.9 he bark of K. gitingensis yielded a complex mixture of alkaloids, including paravallarine, N-methylparavallarine, and 20-epiparavallarine.10 Moreover, the stem bark of the plant was reported to contain lanitine (2α-hydroxyN-methylparavallarine) and its 2β-isomer.11 his study is part of our research on the chemical constituents and bioactivities of plants endemic and native to the Philippines. We recently reported the isolation and identiication of ursolic acid (1), squalene (2), a mixture of α-amyrin acetate (3a) and lupeol acetate (3b) from the leaves, and 1-3 and isoscopoletin (4) from the twigs, of K. gitingensis (Figure 1).12 We report herein the results of the cytotoxicity studies on 1-4 from the leaves and twigs of K. gitingensis. MATERIALS AND METHODS Sample Collection Samples of leaves and twigs of Kibatalia gitingensis (Elm.) Woodson were collected from the De La Salle University–Science and Technology Complex (DLSU-STC) reforested area in February 2014. he samples were authenticated and deposited at the De La Salle University Herbarium with voucher specimen #908. Isolation and Structure Elucidation he isolation and structure elucidation of 1-4 from the leaves and twigs of K. gitingensis were reported previously.12 Preparation of Compounds for Cytotoxicity Tests he compounds (1-4) from K. gitingensis were dissolved in dimethyl sulfoxide (DMSO) to make a 4 mg/mL stock solution. Working solutions were prepared in complete growth medium to a inal non-toxic DMSO concentration of 0.1%. Cite this article: De Los Reyes M M, Oyong G G, Antonio S. Ng V, Shen C C, Ragasa C Y. Cytotoxic Compounds from Kibatalia gitingensis (Elm.) Woodson Pharmacognosy Journal. 2017;9(1):1-7. 8 Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017 De Los Reyes et al.: Cytotoxicity of K. gitingensis 0.39 µg/mL. Wells with no compound served as negative controls, wells with ZeocinTM (Gibco®, USA) served as positive controls, and wells containing only cell culture media were used to correct for background color. he cells were further incubated (37°C, 5% CO2, 98% humidity) for 4 days. Ten microliters of PrestoBlue® was added to each well. he cells were incubated (37°C, 5% CO2, 98% humidity) for 2 hr. Absorbance measurements were carried out using the BioTek ELx800 Absorbance Microplate Reader (BioTek® Instruments, Inc., U.S.A.) at 570 nm and normalized to 600 nm values (reference wavelength). Absorbance readings were used to calculate for the cell viability for each sample concentration following the equation below. Cell viability (%) = Figure 1: Chemical structures of ursolic acid (1), squalene (2), α-amyrin acetate (3a), lupeol acetate (3b), and isoscopoletin (4) from Kibatalia gitingensis. Maintenance and Preparation of Cells for Cytotoxicity Tests he cytotoxicity of 1-4 from the dichloromethane (CH2Cl2) extracts of K. gitingensis was tested on the following human cell lines: breast cancer (MCF-7) and colon cancer (HCT-116 and HT-29) (ATCC, Manassas, Virginia, U.S.A.), and human dermal ibroblast-neonatal (HDFn; Invitrogen Life Technologies, U.S.A.), which are routinely maintained at the Cell and Tissue Culture Laboratory, Molecular Science Unit, Center for Natural Science and Environmental Research, De La Salle University, Manila, Philippines. Following standard procedures,13,14 cells were grown in Dulbecco’s Modiied Eagle Medium (DMEM, Gibco®, USA) containing 10% fetal bovine serum (FBS, Gibco®, USA) and 1x antibiotic-antimycotic (Gibco®, USA) and kept in an incubator (37°C, 5% CO2, 98% humidity). At 80% conluence, the monolayers were washed with phosphate-bufered saline (PBS, pH 7.4, Gibco®, USA), trypsinized with 0.05% Trypsin-EDTA (Gibco®, USA), and resuspended with fresh complete media. Cells were counted following standard trypan blue exclusion method using 0.4% Trypan Blue Solution (Gibco®, USA). Cells were seeded in 100-µL aliquots into a 96-well microtiter plate (FalconTM, USA) using a inal inoculation density of 1 × 104 cells/well. he plates were further incubated overnight (37°C, 5% CO2, 98% humidity) until complete cell attachment was reached. hese cells were used for the cytotoxicity studies as described below. Cell Viability Assay he cytotoxicity of the K. gitingensis compounds was determined in an in vitro cell viability assay using PrestoBlue® (Molecular Probes®, Invitrogen, USA). his test is based on the principle that the enzyme, mitochondrial reductase, present in viable cells, can reduce the nonluorescent, blue resazurin dye in the reagent, converting it to resoruin which is red and highly luorescent. Hence, only viable cells are able to cause color change. he conversion is proportional to the number of metabolically active cells and is correlated to absorbance measurements. To the monolayers in the microtiter plate, 100 µL of ilter-sterilized 1-4 were added to corresponding wells at two-fold serial dilutions to make inal screening concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017 (Absorbance of Treated sample − Absorbance of Blank) (Absorbance of Negative Control − Absorbance of Blank) × 100 Nonlinear regression and statistical analyses were done using GraphPad Prism 7.00 (GraphPad Sotware, Inc.) to extrapolate the half maximal inhibitory concentration, IC50, the concentration of the compound which resulted in a 50% reduction in cell viability. he cytotoxicity of 1-4 was expressed as IC50 values. All tests were performed in triplicates and data were shown as means. he extra sum-of-squares F test was used to evaluate the diferences in the best-it parameter (half maximal inhibitory concentration) among data sets (treatments) and to determine the diferences among dose-response curve its according to the sotware’s recommended approach. One-way ANOVA (p<0.05) was also used to determine signiicant diferences among treatments, followed by Tukey’s multiple comparison post hoc test (p<0.05), to compare diferent pairs of data sets. Results were considered signiicant at p<0.05. RESULTS AND DISCUSION Ursolic acid (1), squalene (2), a mixture of α-amyrin acetate (3a) and lupeol acetate (3b), and isoscopoletin (4), isolated from the dichloromethane extracts of the leaves and twigs of K. gitingensis, were evaluated for their anti-proliferative activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal ibroblast-neonatal (HDFn), using the in vitro PrestoBlue® cell viability assay. he % cell viability as a function of the logarithmic values of compound concentration is shown in Figures 2 and 3. Most plots nearly follow the typical sigmoidal curve which is characteristic of an inhibitory doseresponse relationship between treatments and cell viability. Figure 2 compares the anti-proliferative efects per cell line, while Figure 3 compares the efects per compound. he corresponding IC50 values are summarized in Table 1. he breast cancer cell line (MCF-7) is only moderately susceptible to 1 and 3a and 3b, with IC50 values of 8.642 and 11.13 μg/mL, respectively, and least susceptible to 2 and 4 with IC50 values of 25.87 and 23.35 μg/mL. One-way ANOVA showed statistical diference between treatments (p<0.0001), but Tukey’s multiple comparison post hoc test revealed that there are no pairwise diferences between 1 and 3a and 3b, and 2 and 4 (p>0.05). he colon cancer cell line (HCT-116) is most susceptible to 4 with IC50 values of 4.065 μg/mL, but showed moderate susceptibility only to 1, 2, and 3a and 3b, with IC50 values of 7.225, 9.226, and 11.09 μg/mL, respectively. One-way ANOVA showed that all treatments are statistically diferent (p<0.0001), but Tukey’s multiple comparison post hoc test showed no pairwise diferences between 1 and 2, and 2 and 3a and 3b (p>0.05). he growth of the other colon cancer cell line (HT-29) exhibited 9 De Los Reyes et al.: Cytotoxicity of K. gitingensis Figure 2: Cytotoxic activities of 1-4 and Zeocin (per cell line). Extra sum-of-squares F test was performed to evaluate diferences in: (A) best-it parameters (IC50) among treatments, (B) dose-response curve its. Results: MCF-7 (A) F(DFn, DFd) = F(5,124) = 4.398, p=0.0010 and (B) F(10,124) = 8.142, p<0.0001; HCT-116 (A) F(5,123) = 4.477, p=0.0009 and (B) F(10,123) = 2.513, p=0.0087; HT-29 (A) F(5,124) = 3.419, p=0.0063 and (B) F(10,124) = 2.221, p=0.0205; HDFn (A) F(5,124) = 2.62, p=0.0274 and (B) F(10,124) = 1.888, p=0.0528. Figure 3: Cytotoxic activities of 1-4 (per compound). Extra sum-of-squares F test was performed to evaluate diferences in: (A) best-it parameters (IC50) among treatments, (B) dose-response curve its. Results: 1 (A) F(DFn,DFd) = F(3,88) = 5.473, p=0.0017, (B) F(6,88) = 3.085, p=0.0087; 2 (A) F(3,88) = 25.03, p<0.0001, (B) F(6,88) = 14.24, p<0.0001; 3a and 3b (A) F(3,88) = 8.594, p<0.0001, (B) F(6,88) = 4.419, p=0.0006; 4 (A) F(3,88) = 15.19, p<0.0001, (B) F(6,88) = 7.728, p<0.0001. 10 Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017 De Los Reyes et al.: Cytotoxicity of K. gitingensis Table 1: Cytotoxic activities (IC50) of 1-4 and Zeocin against MCF-7, HCT-116, HT-29 and HDFn Sample 1 IC50* (µg/mL) MCF-7 HCT-116 HT-29 HDFn 8.642 7.225 0.8836 6.218 2 25.87 9.226 0.6931 5.519 3a and 3b 11.13 11.09 1.083 7.628 4 23.35 4.065 1.054 2.106 Zeocin 4.168 1.856 1.318 2.713 *IC50 values were extrapolated from dose-response curves generated from nonlinear regression analysis done using GraphPad Prism 7.00. For each cell line, one-way ANOVA was conducted to determine diferences between data sets (treatments). he results are: MCF-7, F(5, 118) = 31.17, p < 0.0001; HCT-116, F(5, 117) = 84.93, p < 0.0001; HT-29, F(5, 118) = 65.87, p < 0.0001; HDFn, F(5, 118) = 51.73, p < 0.0001. Results of the Tukey’s multiple comparison post hoc test are discussed in this section. the strongest inhibition at the lowest concentrations of the compounds, with IC50 values of 0.6931, 0.8836, 1.054, and 1.083 μg/mL for 2, 1, 4, and 3a and 3b, respectively. Tukey’s multiple comparison post hoc test showed statistical diferences between 2 and all other samples except 1 (p>0.05). he normal cell line, HDFn, responded to all the compounds, with IC50values of 2.106, 5.519, 6.218, and 7.628 μg/mL for 4, 2, 1, and 3a and 3b, respectively. he pairs of compounds, 1 and 2, and 1 and 3a and 3b are not statistically diferent (p>0.05). All the cell lines are susceptible to Zeocin. Data analysis showed statistical diferences in the best-it parameter (half maximal inhibitory concentration) among treatments and among the dose-response curve its (Figures 2 and 3). Overall, comparing the three human cancer cell lines, HT-29 showed the most cytotoxic response with comparable IC50 values for all the compounds tested. his was followed by HCT-116 cells which was most afected by 4 with an IC50 value of 4.065. Among the cancer cell lines tested, MCF-7 showed the least response to the compounds. The compounds exhibited cytotoxic activities against the normal cell line, HDFn. he known anti-cancer drug, Zeocin, showed anti-proliferative activities as expected. Overall, 1-4 showed varying, but promising cytotoxic properties, especially for the treatment of HT-29 type of colon cancer cells. he US National Cancer Institute has deined the active cytotoxic limits of natural products as 20 μg/mL or less for crude extracts and 4 μg/mL or less for pure compounds.15 Pure compounds that exhibit active cytotoxicity may have some potential for drug development.14 he results showed that 1-4 from the dichloromethane extracts of K. gitingensis leaves and twigs can be further evaluated for the treatment especially of human colorectal type of cancer. he study also revealed that the cytotoxic activities of 1-4 were a function of the speciic type of cancer cells targeted. When the two colon cancer cell lines were compared, the IC50 values of 1-4 for HT-29 were lower than HCT-116, implying that the former could be more susceptible to anticancer treatments using the compounds tested. A diference in treatment responses between two colon cancer cell lines was also seen in previous studies.16,17 It was reported that changes in the expression proiles of genes associated with drug sensitivity between HCT-116 and HT-29 could inluence how the cells react to diferent inhibitory compounds.18 A similar study using four human colon cancer cell lines (HCT-116, HT-29, HCT-15, and KM-12) showed that gene expression proiling ater inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, a known inhibitor of the hsp90 molecular chaperone, could explain the cells’ response under diferent treatment parameters.19 Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017 Previous studies revealed that ursolic acid (1), squalene (2), α-amyrin acetate (3a) lupeol acetate (3b), and isoscopoletin (4) exhibited cytotoxic properties. Ursolic acid (1) was reported to promote apoptosis in tumor cells by activation of caspases and modulation of pathways inluencing cell proliferation and migration.20 It also decreased growth and induced apoptosis in gastric cancer cell line BGC-803 and hepatocellular cancer cell H22 xenograt, both in vivo and in vitro studies.21 Other works showed that 1 exhibited anti-tumor activity against human colon carcinoma HCT15 cells,22 and inhibited the colon cancer-initiating cells by targeting STAT3.23 Triterpene 1 and betulinic acid were found useful as therapeutic agents against estrogen-dependent tumors.24 Furthermore, the antiproliferative and apoptotic efects of 1 was found to have potential therapeutic use against prostate cancer.25 A recent study reported that 1 suppressed the proliferation of Jurkat leukemic T-cells, inhibiting PMA/PHA induced IL-2 and TNF-α production in a concentration- and time-dependent manner.26 Another study using cervical cancer TC-1 cells reported that ursolic acid-activated autophagy induced cytotoxicity and reduced tumor growth in a concentration-dependent manner as well.27 he anti-tumor activities of 1 against U87MG brain cancer cells were evaluated and it was found that both G1-phase arrest and autophagy were induced by the compound.28 In a study evaluating the anti-cancer properties of ursolic acid and three lavonoids, daidzein, baicalein, and hesperidin, it was found that 1 and baicalein inhibited the proliferation of MCF-7 breast cancer cells induced by PhIP, a food-derived carcinogen exhibiting estrogenic activities.29 he anti-cancer potential of 1 present in diferent berries has been reviewed.20 hus, ursolic acid (1) was reported to exhibit cytotoxic properties against diferent cancer cells including colon and breast cancer cell lines which corroborate our indings that 1 showed high cytotoxicity against colon cancer cells with the lowest IC50 values of 0.8836 μg/mL obtained for HT-29, 7.225 μg/mL for HCT-116, and 8.642 μg/mL for MCF-7. Squalene (2) was reported to exhibit anti-tumor activities against colon cancer in rodents.30 It also reduced colonic aberrant crypt foci (ACF) formation and crypt multiplicity in laboratory rats, indicating potential chemopreventive activities against colon carcinogenesis.31 In a study evaluating the anti-proliferative efects of squalene and other compounds from palm oil against two human breast cancer cell lines, MDA-MB-231 and MCF-7, it was found that there was a suppression of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) in breast cancer cells exposed briely to tumor necrosis factor-alpha (TNF-α),32,33 hence afecting the mechanisms of apoptosis and carcinogenesis. he protective and therapeutic efects of squalene-containing compounds on skin tumor cells in laboratory mice have been reported as well.34 Relevant reviews on the bioactive properties of squalene have also been provided.35,36 hus, 2 was reported to exhibit cytotoxic properties against colon and breast cancer cells which corroborate our indings that 2 showed high to moderate cytotoxicities against colon cancer cells, HT-29 and HCT-116, and breast cancer cells, MCF-7, with IC50 values of 0.6931, 9.226, and 25.87 μg/mL, respectively. α-Amyrin acetate (3a) was mostly studied for its various potential medicinal applications. At a concentration of 100 mg/kg, 3a isolated from Alstonia boonei showed signiicant (p<0.05) inhibition of egg albumen-induced paw edema in mice.37 he same study showed that it promoted 60.3% reduction in total leucocyte count and signiicant (p<0.05) suppression (47.9%) of neutrophil iniltration. Lupeol, lupeol acetate and α-amyrin acetate exhibited signiicant anti-tyrosinase activity against the mushroom tyrosinase enzyme, with percent inhibitions of 67.7%, 66.2% and 62.2%, respectively,38 indicating potential melanin biosynthesis inhibitory properties. Both α-amyrin acetate and β-amyrin acetate were also reported to exhibit sedative, anxiolytic 11 De Los Reyes et al.: Cytotoxicity of K. gitingensis and anti-convulsant properties.39 Very few studies have been made on the cytotoxic properties of 3a. he dichloromethane extract of Ficus odorata (Blanco) Merr., containing α-amyrin acetate, 1-sitosteryl-3-βglucopyranoside-6’-O-palmitate, squalene, lutein, lupeol acetate, and β-carotene, exhibited anti-proliferative activities against the human cancer cells, lung adenocarcinoma epithelial (A549), stomach adenocarcinoma (AGS), prostate (PC3), and colon adenocarcinoma (HT29).40 Lupeol acetate (3b) was reported to exhibit cytotoxic activity against breast cancer cell (MCF7) with an IC50 value of 26 µg/mL.41 Triterpenes, germincol, lupeol, α-amyrin, β-amyrin, olean-18-ene, and lupeol acetate, were isolated from the methanol extract of leaves and stems of Lactuca steriolla and showed varying cytotoxic activities against non-small cell lung adenocarcinoma cells (A549), human hepatocellular liver carcinoma cells (HepG2), human breast carcinoma cells (MCF7) and human colon carcinoma cells (HCT116).42 he chloroform extract of the leaves of Acokanthera oblongifolia, containing mixtures of isolated triterpenes, α-amyrin, lupeol acetate, lupeol, betulinaldehyde, and betulinic acid, showed some cytotoxic activities against human cancer cell lines, hepatocellular carcinoma (HepG2), breast adenocarcinoma (MCF7) and colorectal (HCT116), with IC50 values of 37.6, 65.4 and 66.8 μg/ml, respectively.43 hus, 3a and 3b were reported to exhibit cytotoxic properties against colon and breast cancer cells which corroborate our indings that the mixture of 3a and 3b showed high to moderate cytotoxic properties against colon cancer cells, HT-29 and HCT-116, and breast cancer cells, MCF-7, with IC50 values of 1.083, 11.09, and 11.13 μg/mL, respectively. It is hypothesized that the synergistic efects of both compounds could have caused the observed anti-proliferative efects against the cancer cells studied. Isoscopoletin (4) showed substantial inhibition in a cell proliferation assay using human CCRF-CEM leukaemia cells with an IC50 value of 4.0 µM.44 Another study reported that 4 exhibited cytotoxic activities against human lung cancer (A549), human breast cancer cell (MCF7) and human liver cancer (HepG2) with IC50 values of 5.25, 8.58 and 4.76 µM, respectively.45 Moreover, 4 showed cytotoxicity against colon cancer (HCT116) cells with an IC50 value of 10% at 100 ppm.46 Compound 4 from Artemisia argyi, artemisinin from Artemisia annua, and the latter’s semi-synthethic derivative, artesunate, showed the greatest activity in in vitro cytotoxicity tests against HCT116 colon adenocarcinoma cell line, with IC50 values ranging in concentration from micromolar to millimolar amounts.47 It was hypothesized that isoscopoletin enhanced its anti-cancer property by inluencing the activity of p53 tumor protein which is a genetically important process in cancer progression. hus, 4 was reported to exhibit cytotoxic properties against several cancer cell lines such as colon and breast which corroborate our indings that 4 showed varying cytotoxic activities against colon cancer cells, HT-29 and HCT-116, and breast cancer cells, MCF-7, with IC50 values of 1.054, 4.065, and 23.35 μg/mL, respectively. Compound 4 also exhibited the highest cytotoxicity against the human dermal ibroblast-neonatal (HDFn) normal cell line, with an IC50 value of 2.106 μg/mL. More studies are needed to fully examine and understand the efects of 4 on normal cells. CONCLUSION Ursolic acid (1), squalene (2), a mixture of α-amyrin acetate (3a) and lupeol acetate (3b), and isoscopoletin (4) from the dichloromethane extracts of Kibatalia gitingensis exhibited varying cytotoxic activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a normal cell line, human dermal ibroblast - neonatal (HDFn). he anti-proliferative activities of 1-4 were highest against HT-29, with IC50 values ranging from 0.6931 to 1.083 μg/mL, followed by HCT-116, with IC50 values ranging from 4.065 to 11.09 μg/mL, and 12 MCF-7, with IC50 values ranging from 8.642 to 25.87 μg/mL. Compounds 1-4 were also cytotoxic against HDFn with IC50 values ranging from 2.106 to 7.628 μg/mL. ACKNOWLEDGEMENT A research grant from the De La Salle University Science Foundation, through the University Research Coordination Oice, De La Salle University, Manila, Philippines, is gratefully acknowledged. Plant samples were authenticated by Dr. Emelina H. Mandia of the Biology Department, De La Salle University. CONFLICT OF INTEREST here is no conlict of interest. REFERENCES 1. World Conservation Monitoring Centre. 1998. Kibatalia gitingensis. The IUCN Red List of Threatened Species 1998: e.T37341A10045636. http://dx.doi. org/10.2305/IUCN.UK.1998.RLTS.T37341A10045636.en. 2. Dayan Md P, Reaviles RS, Bandian Indigenous DB. Forest Tree Species in Laguna Province. DENR Recommends. 2006;5a:7-8. 3. Aguilar-Santos G. Gitingensine, a new alkaloid from the leaves of Kibatalia gitingensis. Philipp J Sci. 1965;94:217-20. 4. Aguilar-Santos G, Santos E, Crabbé P. Stereo chemistry of the alkaloid gitingensine. 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