|Year : 2018 | Volume
| Issue : 1 | Page : 9-15
Cytotoxic Compounds from Wrightia pubescens (R.Br.)
Mariquit M De Los Reyes1, Glenn G Oyong2, Vincent Antonio S. Ng3, Chien-Chang Shen4, Consolacion Y Ragasa5
1 Biology Department, De La Salle University Laguna Campus, Biñan City, Laguna 4024; De La Salle University, 2401 Taft Avenue, Manila 0922, Philippines
2 Biology Department; Center for Natural Science and Environmental Research, De La Salle University, 2401 Taft Avenue, Manila 0922, Philippines
3 Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 0922, Philippines
4 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan
5 Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 0922; De La Salle University Laguna Campus, Biñan City, Laguna 4024, Philippines
|Date of Web Publication||19-Feb-2018|
Dr. Mariquit M De Los Reyes
Biology Department, De La Salle University, 2401 aft Avenue, Manila 0922
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Mixtures of ursolic acid (1) and oleanolic acid (2) (1:1 and 1:2), oleanolic acid (2), squalene (3), chlorophyll a (4), wrightiadione (5), and α-amyrin acetate (6) were isolated from the dichloromethane (CH2Cl2) extracts of the leaves and twigs of Wrightia pubescens (R.Br.). Objectives: To test for the cytotoxicity potentials of 1–6. Materials and Methods: The antiproliferative activities of 1–6 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), were evaluated using the PrestoBlue® cell viability assay. Results: Compounds 4, 1 and 2 (1:2), 2, 1 and 2 (1:1), and 5 exhibited the most cytotoxic effects against HT-29 with half maximal inhibitory concentration (IC50) values of 0.68, 0.74, 0.89, 1.70, and 4.07 μg/mL, respectively. Comparing 2 with its 1:1 mixture with 1 (IC50 = 1.70 and 7.18 μg/mL for HT-29 and HCT-116, respectively) and 1:2 mixture with 1 (0.74 and 3.46 μg/mL for HT-29 and HCT-116, respectively), 2 also showed strong cytotoxic potential against HT-29 and HCT-116 (0.89 and 2.33 μg/mL, respectively). Unlike the mixtures which exhibited low effects on MCF-7 (IC50 = 20.75 and 30.06 μg/mL for 1:1 and 1:2, respectively), 2 showed moderate activity against MCF-7 (10.99 μg/mL). Compound 6 showed the highest cytotoxicity against HCT-116 (IC50 = 4.07 μg/mL). Conclusion: Mixtures of 1 and 2 (1:1 and 1:2), 2, 3, 4, 5, and 6 from the CH2Cl2extracts of the leaves and twigs of W. pubescens (R.Br.) exhibited varying cytotoxic activities. All the compounds except 6 exhibited the strongest cytotoxic effects against HT-29. On the other hand, 6 was most cytotoxic against HCT-116. Overall, the toxicities of 1–6 were highest against HT-29, followed by HCT-116 and MCF-7. All the compounds showed varying activities against HDFn (IC50 <30 μg/mL).
Abbreviation Used: IC50: Half maximal inhibitory concentration.
Keywords: Apocynaceae, chlorophyll a, cytotoxicity, half maximal inhibitory concentration, HCT-116, HT-29, HDFn, MCF-7, oleanolic acid, PrestoBlue® cell viability assay, squalene, ursolic acid, Wrightia pubescens R. Brown, wrightiadione, α-amyrin acetate
|How to cite this article:|
De Los Reyes MM, Oyong GG, S. Ng VA, Shen CC, Ragasa CY. Cytotoxic Compounds from Wrightia pubescens (R.Br.). Phcog Res 2018;10:9-15
|How to cite this URL:|
De Los Reyes MM, Oyong GG, S. Ng VA, Shen CC, Ragasa CY. Cytotoxic Compounds from Wrightia pubescens (R.Br.). Phcog Res [serial online] 2018 [cited 2018 Sep 19];10:9-15. Available from: http://www.phcogres.com/text.asp?2018/10/1/9/225816
Abbreviation Used: IC50: Half maximal inhibitory concentration.
Mixtures of ursolic acid (1) and oleanolic acid (2) (1:1 and 1:2), oleanolic acid (2), squalene (3), chlorophyll a (4), wrightiadione (5), and α-amyrin acetate (6), isolated from the dichloromethane extracts of the leaves and twigs of Wrightia pubescens (R.Br.), showed 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 fibroblast-neonatal (HDFn), as evaluated using the PrestoBlue® cell viability assay.
| Introduction|| |
Wrightia pubescens (R. Br.), of the family Apocynaceae, is one of the eight known species of Wrightia in Malaysia. Locally found in the Philippines where it is known as “lanete,” it is also abundant in mainland China, India, and Australia. W. pubescens is a medium-sized to fairly-large tree which can grow up to 35-m tall in deciduous lowland thickets and forests., The wood is normally used to make furniture, paper, pencil, and musical instruments. In traditional medicine, the root and bark extracts from the tree are employed to treat scrofula and rheumatic arthralgia, and the latex is used against dysentery. In Chinese medicine, preparations containing W. pubescens are used to treat acute upper respiratory infection in children, intractable hiccups,, and osteoarthritis. The plant's latex has been shown to exhibit inhibitory activities on prostaglandin E2 production and cyclooxygenase 2 protein expression in RAW 264.7 mouse macrophages, and these were associated to the anti-inflammatory and antinociceptive properties of the plant.
This study is part of our research on the chemical constituents and bioactivities of trees found at the riparian forest and reforested area of De La Salle University Laguna Campus, Laguna, Philippines. The other trees studied included Calophyllum inophyllum Linn.,Cordia dichotoma G. Forst,Dysoxylum gaudichaudianum (A. Juss.) Miq.,,Kibatalia gitingensis (Elm.) Woodson,, and Pipturus arborescens (Link) C. B. Rob., Studies on the chemical constituents and cytotoxic properties of compounds isolated from the dichloromethane (CH2 Cl2) extracts of these plants have been reported previously.,,,,,,, In an earlier study on W. pubescens from the same site, the isolation and identification of ursolic acid, oleanolic acid, squalene, β-sitosterol, and chlorophyll a from the leaves; and ursolic acid, oleanolic acid, α-amyrin acetate, and wrightiadione from the twigs were reported [Figure 1]., We report herein the results of the cytotoxicity studies on the following compounds from the leaves and twigs of W. pubescens: ursolic acid (1) and oleanolic acid (2) in a 1:1 ratio, and 1 and 2 in a 1:2ratio, (2), squalene (3), chlorophyll a (4), wrightiadione (5), and α-amyrin acetate (6).
|Figure 1: Chemical structures of ursolic acid (1), oleanolic acid (2), squalene (3), chlorophyll a (4), wrightiadione (5), and a-amyrin acetate (6) from Wrightia pubescens|
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| Materials and Methods|| |
Samples of leaves and twigs of W. pubescens (R. Br.) were collected from the DLSU Laguna Campus riparian forest in February 2014. The samples were authenticated previously and deposited at DLSU with collection number #915.
Isolation and structure elucidation
The isolation and structure elucidation of 1–6 from the leaves and twigs of W. pubescens were reported previously.,
Preparation of compounds for cytotoxicity tests
The compounds (1–6) from W. pubescens were dissolved in dimethyl sulfoxide (DMSO) to make a 4 mg/mL stock solution. Working solutions were prepared in complete growth medium to a final nontoxic DMSO concentration of 0.1%.
Maintenance and preparation of cell lines for cytotoxicity tests
The effects on the cell proliferation of 1–6 from the dichloromethane extracts of W. pubescens were tested on the following human cell lines: breast cancer (MCF-7) and colon cancer (HCT-116 and HT-29) (ATCC, Manassas, Virginia, USA), and human dermal fibroblast-neonatal (HDFn; Invitrogen Life Technologies, USA), 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,, cells were grown in Dulbecco's Modified Eagle Medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1X antibiotic-antimycotic (Gibco, USA) and kept in an incubator (37°C, 5% CO2, 98% humidity). At about 80% confluence, the monolayers were prepared for cell counting and inoculation. The cells were washed with phosphate-buffered saline (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 (Falcon, USA) using a final inoculation density of 1 × 104 viable cells/well. The plates were further incubated overnight (37°C, 5% CO2, 98% humidity) until cell attachment was achieved. These monolayer cultures were used for the cytotoxicity studies as described below.
Cell viability assay
The cytotoxicity of the W. pubescens compounds was determined in an in vitro cell viability test using PrestoBlue (Molecular Probes, Invitrogen, USA). This bioassay is based on the ability of viable cells with active enzymes, mitochondrial reductases of the electron transport chain, to convert the resazurin dye (blue and nonfluorescent) to resorufin (red and highly fluorescent). The conversion is proportional to the number of metabolically active cells and is determined quantitatively using absorbance or fluorescence measurements. To the monolayers in the microtiter plate, 100 μL of filter – sterilized 1–6 were added to corresponding wells at two-fold serial dilutions to make final screening concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0.39 μg/mL, respectively. Wells with no compound added served as negative controls, wells with Zeocin™ (Gibco, USA) served as positive controls, and wells containing only cell culture media were used to correct for background color. The cells were further incubated (37°C, 5% CO2, 98% humidity) for 4 days. Ten microliters of PrestoBlue® was added to each well. The cells were further incubated (37°C, 5% CO2, 98% humidity) for 2 h. Absorbance was measured using BioTek ELx800 Absorbance Microplate Reader (BioTek Instruments, Inc., USA) at 570 nm and normalized to 600 nm values (reference wavelength). Absorbance readings were used to calculate for the cell viability for each compound concentration following the equation below.
Nonlinear regression and statistical analyses were done using GraphPad Prism 7.02 (GraphPad Software, Inc., USA) to extrapolate the half maximal inhibitory concentration (IC50), the concentration of the compound which resulted in a 50% reduction in cell viability. The cytotoxicity (antiproliferative potential) of 1–6 was expressed as IC50 values. All tests were performed in triplicates, and data were shown as means ± standard error of mean. The extra sum-of-squares F-test was used to evaluate the differences in the best-fit parameter (half maximal inhibitory concentration) among data sets (treatments) and to determine the differences among dose-response curve fits following the software's manual. One-way ANOVA was used to determine differences in IC50 under different treatments, followed by Tukey's multiple comparison post hoc test to further evaluate differences between pairs of data. The results were considered significant at P< 0.05.
| Results and Discussion|| |
Ursolic acid (1) and oleanolic acid (2) (1:1), 1 and 2 (1:2), 2, squalene (3), chlorophyll a (4), wrightiadione (5), and α-amyrin acetate (6), isolated from the dichloromethane extracts of the leaves and twigs of W. pubescens, were evaluated for their antiproliferative activities against three human cancer cell lines, breast (MCF-7) and colon (HT-29 and HCT-116), and a human normal cell line, HDFn, using the in vitro PrestoBlue cell viability assay. Zeocin, a known anticancer drug, was used as positive control. The percentage cell viability as a function of the logarithmic values of compound concentration is shown in [Figure 2] and [Figure 3]. Most of the curves follow the typical sigmoidal curve which is characteristic of an inhibitory dose-response relationship between treatments and cell viability. [Figure 2] shows the antiproliferative effects per cell line, whereas [Figure 3] shows the effects per treatment used. The IC50 values are summarized in [Table 1].
|Figure 2: Cytotoxicity of 1–6 (per cell line). Extra sum-of-squares F-test was performed to evaluate differences in (a) best-fit parameter (half maximal inhibitory concentration) among treatments, and (b) dose-response curve fits. Results: MCF-7 (a) F (DFn, DFd) = F (8, 190) = 5.522 (P < 0.0001), (b) F (16, 190) = 6.688 (P < 0.0001); HCT-116 (a) F (8, 182) = 21.81 (P < 0.0001), (b) F (16, 182) = 11.29 (P < 0.0001); HT-29 (a) F (8, 190) = 7.503 (P < 0.0001), (b) F (16, 190) = 6.09 (P < 0.0001); HDFn (a) F (8, 190) = 3.745 (P = 0.0004), (b) F (16, 190) = 2.721 (P = 0.0006)|
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|Figure 3: Cytotoxicity of 1–6 (per sample). Extra sum-of-squares F test for (a) best-fit parameters (half maximal inhibitory concentration) and (b) dose-response curve fits. Results: 1 and 2 (1:1) (a) F (DFn, DFd) = F (3, 80) = 5.96 (P = 0.0010), (b) F (6, 80) = 3.364 (P = 0.0052); 1 and 2 (1:2) (a) F (3, 88) = 23.5 (P < 0.0001), (b) F (6, 88) = 13.38 (P < 0.0001); 2 (a) F (3, 88) =19.03 (P < 0.0001), (b) F (6, 88) =10.22 (P < 0.0001); 3 (a) F (3, 88) =1.465 (P = 0.2297), (b) F (6, 88) = 2.843 (P = 0.0141); 4 (a) F (3, 88) = 23.08 (P < 0.0001), (b) F (6, 88) =12.48 (P < 0.0001); 5 (a) F (3, 88) =11.08 (P < 0.0001), (b) F (6, 88) = 7.632 (P < 0.0001); 6 (a) F (3, 88) = 2.929 (P = 0.0380), (b) F (6, 88) = 1.746 (P = 0.1198)|
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|Table 1: Cytotoxic activities (half maximal inhibitory concentration) of 1-6 and Zeocin against MCF-7, HT-29, HCT-116, and HDFn cells|
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The 1:1 mixture of 1 and 2 is strongly effective against HT-29 (IC50 = 1.70 μg/mL), but only moderately effective against HCT-116 (IC50 value = 7.18 μg/mL). The mixture exhibited low toxicity against MCF-7 (IC50 = 20.75 μg/mL). One-way ANOVA showed statistical differences in IC50 values among the four cell lines (P < 0.0001). Tukey's multiple comparison post hoc test revealed significant differences between all pairs of cell lines with P< 0.0001 except between HT-29 and HDFn with P< 0.01. The same trend is seen for the 1:2 mixture of 1 and 2 which is also strongly effective against HT-29 (IC50 = 0.74 μg/mL) and HCT-116 (IC50 = 3.46 μg/mL) but not as effective against MCF-7 (IC50 = 30.06 μg/mL). One-way ANOVA showed statistical differences in IC50 values among the four cell lines (P = 0.0040). However, Tukey's post hoc test showed that there is only significant difference between MCF-7 and HT-29 (P < 0.01). Comparing the overall cytotoxic effects of 1:1 and 1:2 mixtures of 1 and 2 against the cancer cell lines, the data showed that 1:2 is more effective except for MCF-7 cells. Comparing the bioactivities of the two mixtures against 2, the latter exhibited the same trend, with the strongest antiproliferative effect against HT-29 (IC50 = 0.89 μg/mL) and HCT-116 (IC50 = 2.33 μg/mL). Compound 2, however, is more effective against MCF-7 (IC50 = 10.99 μg/mL) compared to its 1:1 and 1:2 mixtures with 1. One-way ANOVA showed statistical differences in IC50 values among the four cell lines (P < 0.0001). Tukey's post hoc test consistently showed significant differences between all pairs of cell lines (P < 0.0001).
Squalene (3) seemed to show the same trend in bioactivity against the cancer cell lines ([Table 1]). However, it failed the test for significance (P = 0.8483) after ANOVA. None of the pair-wise data comparing the cell lines is significant. This implies that though squalene may still be considered bioactive as shown in the previous studies and the magnitude of IC50 values obtained in this work, the significance of this parameter (per cell line) cannot be established statistically. Chlorophyll a (4) exhibited the strongest potential against HT-29 (IC50 = 0.68 μg/mL), but, unlike 1–3, it showed the lowest toxicity against HCT-116 (IC50 = 15.45 μg/mL). One-way ANOVA showed statistical difference between treatments (P < 0.0001), but Tukey's multiple comparison post hoc test revealed that there is no pair-wise difference between MCF-7 and HDFn (P > 0.05). Wrightiadione (5) showed the same trend as 4, exhibiting a strong antiproliferative effect against HT-29 cells (IC50 = 4.07 μg/mL) and MCF-7 cells (IC50 = 5.69 μg/mL), but low toxicity against HCT-116 (IC50 = 25.11 μg/mL). One-way ANOVA showed statistical difference between the treatments (P < 0.0001), but Tukey's multiple comparison post hoc test revealed that there is no pairwise difference between HCT-116 and HDFn (P > 0.05). α-Amyrin acetate (6) is strongly potent for HCT-116 (ic50 = 4.07 μg/ml) and moderately toxic against HT-29 (IC50 = 7.97 μg/mL) and MCF-7 (IC50 = 13.81 μg/mL). One-way ANOVA showed statistical difference between treatments (P < 0.0001), but Tukey's multiple comparison post hoc test revealed that there is no pairwise difference between HT-29 and HDFn (P > 0.05).
When the two colon cancer cell lines (HCT-116 and HT-29) are compared, the IC50 values of 1–6 for HT-29 were generally lower, implying that this cell line is more responsive to anticancer treatments using the samples tested. Differences in treatment response between the same colon cancer cell lines were also seen in previous studies.,, It was reported that differences in the expression profiles of genes associated with drug sensitivity in HCT-116 and HT-29 cells could be a contributory factor influencing how the cells respond to inhibitory compounds. In another work, variations in the sensitivity of HCT-116 and HT-29 cells against two known metabolic stressor compounds, ribavirin and metformin, were attributed to the genetic and metabolic activities of the cell lines, suggesting differences in the use of nutrients and the metabolic pathways taken which then influence in vitro survival under stressors. Another study which evaluated four human colon cancer cells (HCT-116, HT-29, HCT-15, and KM-12) showed that gene expression profiling following the inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone, could explain why cellular response to similar treatment conditions varied.
For all the samples tested, strong to low antiproliferative activities against the normal cell line, HDFn, was seen. As discussed above, the IC50 value of 0.10 μg/mL for 3 cannot be claimed as determined statistically. Hence, from the remaining data, the strongest inhibition was seen in the 1:1 mixture of 1 and 2 with an IC50 value of 2.92 μg/mL. Zeocin, as expected, showed strong cytotoxicity against all the cell lines used (strongest against HT-29 with IC50 = 1.32 μg/mL). ANOVA and Tukey's post hoc analyses consistently showed that the IC50 values are significant (P < 0.0001).Overall, 1 and 2 (1:1), 1 and 2 (1:2), 2, 3, 4, 5 and 6 exhibited the strongest antiproliferative effects against the HT-29 cells, followed by HCT-116. Both are colon cancer cell lines. Among the cancer cell lines tested, MCF-7 showed the least response to the samples. The samples also exhibited cytotoxic activities against the normal cell line, HDFn. The known anticancer drug, Zeocin, showed antiproliferative activities as expected. In general, 1–6 showed varying but promising cytotoxic properties. The US National Cancer Institute has defined 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. Pure compounds that exhibit active cytotoxicity may have some potential for further drug development. The results showed that 1–6, isolated from the dichloromethane extracts of the leaves and twigs of W. pubescens, can be further evaluated for the treatment especially of the human colorectal types of cancer.
Previous studies revealed that ursolic acid (1), oleanolic acid (2), squalene (3), chlorophyll a (4), wrightiadione (5), and α-amyrin acetate (6) exhibited cytotoxic activities.
Ursolic acid (1) was reported to cause apoptosis in tumor cells by activating the enzyme, caspase, which is involved in programmed cell death, and by modulating pathways relevant to cell proliferation and migration. This compound also decreased growth and promoted apoptosis in gastric cancer cell line BGC-803 and hepatocellular cancer cell H22 xenograft, under both in vivo and in vitro studies. Other studies showed that 1 exhibited antitumor activity against human colon carcinoma HCT-15 cells and inhibited colon-cancer-initiating cells by targeting the gene, STAT3, essential in chemical signaling pathways within cells. The triterpenoids 1 and betulinic acid, were found important as therapeutic agents against estrogen-dependent tumors. The cytotoxic activities of 1 against prostate cancer have been reported., Another study showed that 1 suppressed the proliferation of Jurkat leukemic T-cells, inhibiting phorbol myristate acetate/phytohemagglutinin-induced IL-2 and tumor necrosis factor-alpha (TNF-α) in a concentration and time-dependent manner. Another study using TC-1 cervical cancer cells reported that ursolic acid-activated autophagy induced cytotoxicity and reduced tumor growth in a concentration-dependent manner as well. The antitumor activities of 1 against U87MG brain cancer cells were attributed to the G1-phase arrest and autophagy that were both induced by the compound. In a study evaluating the anticancer properties of ursolic acid and the three flavonoids, daidzein, baicalein, and hesperidin, it was found that the mixture of 1 and baicalein inhibited the growth of MCF-7 breast cancer cells which was induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine, a food-derived carcinogen, exhibiting estrogenic activities. Thus, ursolic acid (1) was reported to exhibit cytotoxic properties against different cancer cells which corroborate our findings that 1, in a 1:1 or 1:2 mixture with oleanolic acid (2), showed high cytotoxic activities against colon cancer cells, with the lowest IC50 values obtained for HT-29 (IC50 = 1.70 and 0.74 μg/mL, for the 1:1 and 1:2 ratios of 1 and 2, respectively).
Oleanolic acid (2) was found to be antimutagenic and antitumor, inhibiting the proliferation of gastric, colon, and liver cancer cells by inducing apoptosis and necrosis. Triterpene 2 inhibited mouse skin tumor and exhibited significant antitumor activity against human colon carcinoma cell line HCT-15. Another study identified 2 as an antitumor compound, suppressing aerobic glycolysis in MCF-7 breast cancer cells by promoting a metabolic switch in the PKM2 to PKM1 ratio, an important event in cancer development. An extensive review of the cytotoxic nature of oleanolic acid and other triterpenes has been presented. Thus, oleanolic acid (2) was reported to exhibit cytotoxic properties against different cancer cells which corroborate our findings that pure 2 or in combination with ursolic acid (1) (1:1 or 1:2), showed high cytotoxic activities against colon cancer cells, with the lowest IC50 values obtained for HT-29 (IC50 = 0.89 μg/mL) and HCT-116 (IC50 = 2.33 μg/mL).Squalene (3) was shown to exhibit antitumor activities against colon cancer found in rodents. It also reduced colonic aberrant crypt foci formation and crypt multiplicity in laboratory mice, demonstrating chemopreventive activities against colon carcinogenesis. In a study using compounds extracted from palm oil, squalene and other isolates were found to have antiproliferative effects against two human breast cancer cell lines, MDA-MB-231, and MCF-7, resulting from the suppression of nuclear factor kappa-light-chain-enhancer of activated B-cells in breast cancer cells exposed briefly to TNF-α, hence, affecting apoptosis and carcinogenesis., The protective and therapeutic effects of squalene-containing compounds on skin tumor cells in laboratory mice have been reported as well. Relevant review papers on the bioactivities of squalene and its derivatives have been provided., Thus, 3 was reported to exhibit cytotoxic properties against colon and breast cancer cells which corroborate our findings that 3 generally showed potential antiproliferative activities especially against the colon cancer cells used in the study (IC50= 8.20 μg/mL for HT-29 cells).
Chlorophyll a (4) and its derivatives are popularly used in the traditional medicine for its various therapeutic applications. Natural chlorophyll and its derivatives have been evaluated for wound healing, anti-inflammatory properties, control of calcium oxalate crystals, anticancer activities,,, and chemopreventive effects in humans., A review on the digestion, absorption, and cancer preventive activities of dietary chlorophyll has been presented. In a recent study evaluating the cytotoxic activities of chlorophyll a and its derivatives against human cell lines, it was found that the compounds exhibited photoinduced cytotoxic activities in vitro. Thus, 4 was reported to exhibit anticancer properties which corroborate our findings that 4 showed antiproliferative activities which was very strong against HT-29 colon cancer cells (IC50 = 0.68 μg/mL) and moderately effective against MCF-7 breast cancer cells (IC50 = 8.69 μg/mL).
Wrightiadione (5), isolated from Wrightia tomentosa, was reported to exhibit cytotoxic activity against the murine P-388 lymphocytic leukemia cell line. The activities of 5 were compared with wrightiamines a and b and all were found cytotoxic against the same vincristine-resistant murine leukemia P-388 cells. There are limited reports on the cytotoxic properties of 5. In this study, 5 was found to exhibit antiproliferative activities which was strongest against HT-29 colon cancer cells (IC50 = 4.07 μg/mL) and MCF-7 breast cancer cells (IC50 = 5.69 μg/mL).
α-Amyrin acetate (6) were mostly studied for its various potential medicinal applications. Compound 6, isolated from Alstonia boonei, showed inhibition of egg albumen-induced paw edema in laboratory mice. The same study showed that it promoted reduction in total leukocyte count and suppression of neutrophil infiltration. Lupeol, lupeol acetate, and α-amyrin acetate exhibited anti-tyrosinase activity, indicating potential melanin biosynthesis inhibitory properties. Both α-amyrin acetate and β-amyrin acetate were reported to exhibit sedative, anxiolytic, and anticonvulsant properties. Limited studies have been conducted evaluating the cytotoxic properties of 6 against human cancer cells. The dichloromethane extract of Ficus odorata (Blanco) Merr., containing α-amyrin acetate, 1-sitosteryl-3-β-glucopyranoside-6'-O-palmitate, squalene, lutein, lupeol acetate, and β-carotene, exhibited antiproliferative activities against the human cancer cell lines, lung adenocarcinoma epithelial (A549), stomach adenocarcinoma (AGS), prostate (PC3), and colon adenocarcinoma (HT-29). Thus, 6 was reported to exhibit cytotoxic properties which corroborate our findings that 6 showed antiproliferative activities which was strongest against the two colon cancer cell lines, HCT-116 (IC50 = 4.07 μg/mL) and HT-29 (IC50 = 7.97 μg/mL).
It remains to be explored if other parts of the plant, such as stem bark and roots, will be able to afford the same compounds and exhibit other bioactivities such as antibacterial, anti-inflammatory, and antioxidative, similar to other studies.,,,
| Conclusion|| |
Mixtures of ursolic acid (1) and oleanolic acid (2) (1:1 and 1:2), oleanolic acid (2), squalene (3), chlorophyll a (4), wrightiadione (5), and α-amyrin acetate (6) from the dichloromethane extracts of the leaves and twigs of W. pubescens (R. Br.) 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 fibroblast - neonatal (HDFn) Compounds 4, 1 and 2 (1:2), 2, and 1 and 2 (1:1) exhibited the strongest cytotoxic effects against HT-29 with IC50 values of 0.68, 0.74, 0.89, and 1.70 μg/mL, respectively. The two colon cancer cell lines responded well under all treatments, with HCT-116 generally less susceptible to the treatments. When 2 was compared with its 1:1 mixture with 1 (IC50 = 1.70 and 7.18 μg/mL for HT-29 and HCT-116, respectively) and 1:2 mixture with 1 (IC50 = 0.74 and 3.46 μg/mL for HT-29 and HCT-116, respectively), the data for 2 also showed strong antiproliferative potential against HT-29 (IC50 = 0.89 μg/mL) and HCT-116 (IC50 = 2.33 μg/mL). However, unlike the two mixtures which both exhibited low antiproliferative effects on MCF-7 (IC50 = 20.75 and 30.06 μg/mL for 1:1 and 1:2, respectively), 2 exhibited moderate activity against MCF-7 (IC50 = 10.99 μg/mL). Overall, the activities of 1–6 were highest against HT-29, followed by HCT-116 and MCF-7. Compounds 1–6 also showed varying toxicities against HDFn (IC50 <30 μg/mL).
Financial support and sponsorship
A research grant from the De La Salle University Science Foundation, through the University Research Coordination Office, De La Salle University, Manila, Philippines, is gratefully acknowledged.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Middleton DJ. A revision of Wrightia
in Malesia. Harvard Pap Bot 2005;10:161-82.
Shu DD. Wrightia. In: Flora of China Vol. 16; Wu ZY and Raven PH, eds. Science Press, Beijing, China, and Missouri Botanical Garden Press, St. Louis, Missouri, USA; 1995. p. 174 5.
Van Sam H, Nanthavong K, Kessler PJ. Blumea. Biodivers Evol Biogeogr Plants 2004;49:235.
Song Q, Zhou S. Traditional Chinese medicine enema for treating acute upper respiratory infection of children. Faming Zhuanli Shenqing 2012; CN 102648958 A 20120829.
Liu J, Wang B, Wang B. Chinese medicine for intractable hiccups. Faming Zhuanli Shenqing. 2014; CN 103977156 A 20140813.
Ji Z, Liu Z. Chinese medicine for intractable hiccups. Faming Zhuanli Shenqing. 2014; CN 103705701 A 20140409.
Jiang Y. Osteoarthritis treating plaster manufactured from traditional Chinese medicines. Faming Zhuanli Shenqing. 2012; CN 102697883 A 20121003.
Jittimanee J, Panomket P, Wanrum S. Inhibition of prostaglandin E2 by substances derived from Wrightia pubescens
latex in LPS-activated RAW 264.7 mouse macrophages. J Med Tech Phys Ther 2013;25:36-42.
Ragasa CY, Ebajo VD Jr., De Los Reyes MM, Mandia EH, Brkljača R, Urban S. Triterpenes from Calophyllum inophyllum
Linn. Int J Pharmacogn Phytochem Res 2015;7:718-22.
Ragasa CY, Ebajo VD Jr, Forst G, De Los Reyes MM, Mandia EH, Brkljača R, et al
. Chemical constituents of Cordia dichotoma
G. Forst. J Appl Pharm Sci 2015;5 Suppl 2:16-21.
Ragasa CY, Ng VA, De Los Reyes MM, Mandia EH, Oyong GG, Shen CC. Chemical constituents and cytotoxicity of the leaves of Dysoxylum gaudichaudianum
(A. Juss.) Miq. Pharm Chem 2014;6:182-7.
De Los Reyes MM, Oyong GG, Ng VA, Shen CC, Ragasa CY. Cytotoxic compounds from Dysoxylum gaudichaudianum
(A. Juss.) Miq. Int J Pharmacogn Phytochem Res 2016;8:668-74.
Ragasa CY, Ng VA, De Los Reyes MM, Mandia EH, Shen CC. Triterpenes and a coumarin derivative from Kibatalia gitingensis
(Elm.) Woodson. Pharm Chem 2014;6:360-4.
De Los Reyes MM, Oyong GG, Ng VA, Shen CC, Ragasa CY. Cytotoxic compounds from Kibatalia gitingensis
(Elm.) Woodson. Pharmacogn J 2017;9:8-13.
Ragasa CY, Ng VA, De Los Reyes MM, Mandia EH, Shen CC. Chemical constituents of Pipturus arborescens
. Pharm Lett 2014;6:35-42.
De Los Reyes MM, Oyong GG, Ebajo VD Jr, Ng VA, Shen CC, Ragasa CY. Cytotoxic triterpenes and sterols from Pipturus arborescens
(Link) C.B. Rob. J Appl Pharm Sci 2015;5:23-30.
Ragasa CY, Ng VA, Ebajo V Jr., De Los Reyes MM, Mandia EH, Shen CC. Chemical constituents of Wrightia pubescens
(R. Br.). Pharm Lett 2014;6:14-9.
Ragasa CY, Ng VA, De Los Reyes MM, Mandia EH, Shen CC. An isoflavone from Wrightia pubescens.
Int J Pharmacogn Phytochem Res 2015;7:353-5.
Freshney RI. Culture of Animal Cells: A Manual of Basic Techniques. New York, U.S.A.: Wiley-Liss Inc.; 2000.
Jacinto SD, Chun EA, Montuno AS, Shen CC, Espineli DL, Ragasa CY. Cytotoxic cardenolide and sterols from Calotropis gigantea
. Nat Prod Commun 2011;6:803-6.
Makizumi R, Yang WL, Owen RP, Sharma RR, Ravikumar TS. Alteration of drug sensitivity in human colon cancer cells after exposure to heat: implications for liver metastasis therapy using RFA and chemotherapy. Int J Clin Exp Med 2008;1:117-29.
Richard SM, Martinez Marignac VL. Sensitization to oxaliplatin in HCT116 and HT29 cell lines by metformin and ribavirin and differences in response to mitochondrial glutaminase inhibition. J Cancer Res Ther 2015;11:336-40.
Clarke PA, Hostein I, Banerji U, Stefano FD, Maloney A, Walton M, et al.
Gene expression profiling of human colon cancer cells following inhibition of signal transduction by 17-allylamino-17-demethoxygeldanamycin, an inhibitor of the hsp90 molecular chaperone. Oncogene 2000;19:4125-33.
Geran RI, Greenberg NH, McDonald MM, Schumacher AM, Abbott BJ. Protocols for screening chemical agents and natural products against animal tumour and other biological systems. Cancer Chemother Rep 1972;3:17-9.
Neto CC. Ursolic acid and other pentacyclic triterpenoids anticancer activities and occurrence in berries. In: Stoner GD, Seeram NP, editors. Berries and Cancer Prevention. New York, NY, USA; Springer Science+Business Media LLC, 2011. p. 41-9.
Wang X, Zhang F, Yang L, Mei Y, Long H, Zhang X, et al.
Ursolic acid inhibits proliferation and induces apoptosis of cancer cells in vitro
and in vivo
. J Biomed Biotechnol 2011;2011:419343.
Li J, Guo WJ, Yang QY. Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15. World J Gastroenterol 2002;8:493-5.
Wang W, Zhao C, Jou D, Lü J, Zhang C, Lin L, et al.
Ursolic acid inhibits the growth of colon cancer-initiating cells by targeting STAT3. Anticancer Res 2013;33:4279-84.
Kim HI, Quan FS, Kim JE, Lee NR, Kim HJ, Jo SJ, et al
. Inhibition of estrogen signaling through depletion of estrogen receptor alpha by ursolic acid and betulinic acid from Prunella vulgaris var. lilacina. Biochem Biophys Res Commun 2014;451:282-7.
Kassi E, Papoutsi Z, Pratsinis H, Aligiannis N, Manoussakis M, Moutsatsou P. Ursolic acid, a naturally occurring triterpenoid, demonstrates anticancer activity on human prostate cancer cells. J Cancer Res Clin Oncol 2007;133:493-500.
Shanmugam MK, Ong TH, Kumar AP, Lun CK, Ho PC, Wong PT, et al.
Ursolic acid inhibits the initiation, progression of prostate cancer and prolongs the survival of TRAMP mice by modulating pro-inflammatory pathways. PLoS One 2012;7:e32476.
Kaewthawee N, Brimson S. The effects of ursolic acid on cytokine production via the MPKA pathways in leukemic T-cells. EXCLI J 2013;12:102-14.
Leng S, Hao Y, Du D, Xie S, Hong L, Gu H, et al
. Ursolic acid promotes cancer cell death by inducing Atg5-dependent autophagy. Int J Cancer 2013;133:2781-90.
Shen S, Zhang Y, Zhang R, Tu X, Gong X. Ursolic acid induces autophagy in U87MG cells via ROS-dependent endoplasmic reticulum stress. Chem Biol Interact 2014;218:28-41.
Lee MN, Lee SY, Lee HJ, Seok JH, Lee CJ. Anti-proliferative effects of Daidzein, Baicalein, hesperidin and ursolic acid on human breast cancer cells stimulated by estrogenic compounds. Yakhak Hoechi 2010;54:168-73.
Zhang M, Shen Y. The anti-cancer effects of ursolic acid and oleanolic acid in the digestive system: A review. Shanghai Yiyao 2011;32:606-11.
Oguro T, Liu J, Klaassen CD, Yoshida T. Inhibitory effect of oleanolic acid on 12-O-tetradecanoylphorbol-13-acetate-induced gene expression in mouse skin. Toxicol Sci 1998;45:88-93.
Liu J, Wu N, Ma L, Liu M, Liu G, Zhang Y, et al.
Oleanolic acid suppresses aerobic glycolysis in cancer cells by switching pyruvate kinase type M isoforms. PLoS One 2014;9:e91606.
Chudzik M, Korzonek-Szlacheta I, Król W. Triterpenes as potentially cytotoxic compounds. Molecules 2015;20:1610-25.
Spanova M, Daum G. Squalene-biochemistry, molecular biology, process biotechnology, and applications. Eur J Lipid Sci Technol 2011;113:1299-320.
Rao CV, Newmark HL, Reddy BS. Chemopreventive effect of squalene on colon cancer. Carcinogenesis 1998;19:287-90.
Loganathan R, Radhakrisnan AK, Selvaduray KR, Nesaretnam K. Selective anti-cancer effects of palm phytonutrients on human breast cancer cells. Royal Soc Chem Adv 2015;5:1745-53.
Loganathan R, Selvaduray KR, Nesaretnam K, Radhakrisnan A. Differential and antagonistic effects of palm tocotrienols and other phytonutrients (carotenoids, squalene and coenzyme Q10) on breast cancer cells in vitro
. J Oil Palm Res 2013;25:208-15.
Desai KN, Wei H, Lamartiniere CA. The preventive and therapeutic potential of the squalene-containing compound, Roidex, on tumor promotion and regression. Cancer Lett 1996 19;101:93-6.
Ronco AL, De Stéfani E. Squalene: A multi-task link in the crossroads of cancer and aging. Funct Foods Health Dis 2013;3:462-76.
Edwards BJ. Treatment of chronic leg ulcers with ointment containing soluble chlorophyll. Physiotherapy 1954;40:177-9.
Kephart JC. Chlorophyll derivatives-their chemistry, commercial preparation and uses. Econ Bot 1955;9:3-18.
Larato DC, Pfau FR. Effects of a water-soluble chlorophyllin ointment on gingival inflammation. N Y State Dent J 1970;36:291-3.
Tawashi R, Cousineau M, Sharkawi M. Effect of sodium copper chlorophyllin on the formation of calcium oxalate crystals in rat kidney. Invest Urol 1980;18:90-2
Sternberg ED, Dolphin D, Bruckner C. Porphyrin-based photosensitizers for use in photodynamic therapy. Tetrahedron 1998;54:4151-2.
Nourse WL, Parkhurst RM, Skinner WA, Jordan RT. Photodynamic toxicity of porphyrins and chlorins for a human tumor cell line: Combined light and concentration dose responses for the retained fraction. Biochem Biophys Res Commun 1988;151:506-11.
Henderson BW, Bellnier DA, Greco WR, Sharma A, Pandey RK, Vaughan LA, et al.
An in vivo
quantitative structure-activity relationship for a congeneric series of pyropheophorbide derivatives as photosensitizers for photodynamic therapy. Cancer Res 1997;57:4000-7.
Egner PA, Wang JB, Zhu YR, Zhang BC, Wu Y, Zhang QN, et al.
Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proc Natl Acad Sci U S A 2001 4;98:14601-6.
Egner PA, Muñoz A, Kensler TW. Chemoprevention with chlorophyllin in individuals exposed to dietary aflatoxin. Mutat Res 2003;523-524:209-16.
Hardwick SJ, Carpenter KL, Law NS, Van Der Veen C, Marchant CE, Hird R, et al.
Toxicity of polyunsaturated fatty acid esters for human monocyte-macrophages: The anomalous behaviour of cholesteryl linolenate. Free Radic Res 1997;26:351-62.
Pylina YI, Shadrin DM, Shevchenko OG, Startseva OM, Velegzhaninov IO, Belykh DV, et al.
Dark and photoinduced cytotoxic activity of the new chlorophyll-a derivatives with oligoethylene glycol substituents on the periphery of their macrocycles. Int J Mol Sci 2017;18. pii: E103.
Lin LJ, Topcu G, Lotter H, Ruangrungsi N, Wagner H, Pezzuto JM, et al
. Wrightiadione from Wrightia tomentosa
. Phytochem 1992;31:4333-5.
Hafidh RR, Abas F, Abdulamir AS, Jahanshiri F, Abu Bakar F, Sekawi Z. A review: Cancer research of natural products in Asia. Int J Cancer Res 2009;5:69-82.
Okoye NN, Ajaghaku DL, Okeke HN, Ilodigwe EE, Nworu CS, Okoye FB. Beta-amyrin and alpha-amyrin acetate isolated from the stem bark of Alstonia boonei
display profound anti-inflammatory activity. Pharm Biol 2014;52:1478-86.
Aragão GF, Carneiro LM, Junior AP, Bandeira PN, Lemos IL, Viana GS. Evidence for excitatory and inhibitory amino acids participation in the neuropharmacological activity of alpha- and beta-amyrin acetate. Open Pharmacol J 2009;3:9-16.
Tsai PW, De Castro-Cruz KA, Shen CC, Chiou CT, Ragasa CY. Chemical constituents of Ficus odorata
. Pharm Chem J 2012;46:225-7.
Columba-Palomares MM, Villareal DL, Acevedo Quiroz MM, Marquina Bahena MS, Álvarez Berber DP, Rodríguez-López D. Anti-inflammatory and cytotoxic activities of Bursera copallifera
. Phcogn Mag 2015;11 Suppl S2:322-8. Available from: http://www.phcog.com/text.asp?2015/11/44/322/166067
. [Last accessed on 2017 May 17].
Kabir H, Shah M, Hossain MM, Kabir M, Rahman M, Hasanat A, et al
. Phytochemical screening, antioxidant, thrombolytic, α-amylase inhibition and cytotoxic activities of ethanol extract of Steudnera colocasiifolia
K. Koch leaves. J Young Pharm 2016;8:391-7.
Perveen S, Al-Taweel AM, Fawzy GA, El-Shafae AM, Khan A, Proksch P. Cytotoxic glucosphingolipid from Celtis africana
. Phcog Mag 2015;11 Suppl S1:1-5. Available from: http://www.phcog.com/text.asp?2015/11/42/1/157662
. [Last accessed on 2017 May 17].
Ugur AR, Dagi HT, Ozturk B, Tekin G, Findik D. Assessment of In vitro
antibacterial activity and cytotoxicity effect of Nigella sativa
oil. Phcog Mag 2016;12 Suppl S4:471-4. Available from: http://www.phcog.com/text.asp?2016/12/47/471/191459
. [Last accessed on 2017 May 17].
[Figure 1], [Figure 2], [Figure 3]