Green seaweed Caulerpa racemosa - Chemical constituents, cytotoxicity in breast cancer cells and molecular docking simulation
Grace SangerDjuhria WonggoNurmeilita TaherVerly DotulongAurielle Annalicia SetiawanHappy Kurnia PermatasariSidik MaulanaFahrul NurkolisApollinaire TsopmoBonglee Kim
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Abstract:
Marine and terrestrial organisms are rich in chemical compounds with medicinal and pharmacological properties, including antitumor agents for chemoprevention. Caulerpa racemosa, a marine species, is a potential source of novel compounds with therapeutic agents for human cancer. This study aimed to determine the anticancer activity of C. racemosa extracts in breast cancer cells, identify compounds, and determine the mechanism using computational models. Seaweed (C. racemosa) was taken from North Sulawesi, Indonesia; Followed by authentication and identification according to the previously published protocol and extracted with three different solvent: hexane, ethyl acetate, and ethanol. C. racemosa were evaluated for cytotoxicity against breast cancer MCF-7 cells using MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) assay. Antioxidant activities were assessed based on free radical scavenging (2,2-diphenyl-1-picrylhydrazyl (DPPH)) and ferric-reducing antioxidant power (FRAP) assays. The phytochemical constituents were identified with a liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) system. The interaction of the identified compounds with human epidermal growth factor 2 (HER2) protein was achieved by molecular docking with PyRx-vina application and protein-ligand complex visualization. The hexane extract exhibited the highest cytotoxicity against breast cancer cells, followed by the ethyl acetate and the ethanol extract (IC50 23.7 ± 2.0; 66.7 ± 5.8 and 182.7 ± 14.3 μg/mL). The best antioxidant sample for DPPH was the ethyl acetate extract (IC50 21.5 ± 2.0 μg/mL) while the hexane extract was the most active in the FRAP value (14.5 ± 1.3 μg gallic acid equivalent/g). Data from LC-ESI-QTOF-MS allowed the identification of 21 compounds. The molecular docking study showed that 12 of the compounds could prevent tumors in breast cancer by acting as inhibitors of the anti-apoptotic HER2 protein. C. racemosa has potential in the chemoprevention of breast cancer through its radical scavenging capacity and inhibition of the HER2 protein. More studies are needed to investigate the efficacy of extracts in different models.Keywords:
Phytochemical
MTT assay
The present study evaluated the antioxidant and cytotoxic activities of methanolic extract of aerial parts of Adiantum capillus-veneris L. and its different solvent fractions. The in vitro antioxidant activity was assessed by using 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radicals. The analysis revealed that ethyl acetate soluble fraction had the highest DPPH radicals scavenging property with IC50 value of 1.05 μg/ml as compared to positive control ascorbic acid (IC50 = 1.34 μg/ml). In addition, ex vivo cytotoxicity assay of A. capillus-veneris L. extract and its different fractions were performed against HELA cells line where 5-Fluorouracil was used as positive control. The result demonstrated that ethyl acetate and n-hexane soluble fractions showed prominent cytotoxicity with IC50 value of 5.68 μg/ml and 17.15 μg/ml, respectively. The study affirmed that superior antioxidant and cytotoxic activities were shown by ethyl acetate soluble fraction of methanolic extract of aerial parts of A. capillus-veneris L. growing in Bangladesh which indicate the presence of bioactive phytoconstituents in the extractives.
Dhaka Univ. J. Pharm. Sci. 18(2): 217-222, 2019 (December)
HeLa
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Improvement of MTT assay by 2-chloroadenosine in activation test and cytotoxicity test of lymphocyte
Objective:To sutdy the effect of 2 chloroadenosine(2 ClA),which is specifically cytotoxic to macrophages,on MTT assay in activation test and cytotoxicity test of lymphocyte.Methods:Using cell culture technique,mouse splenic lymphocytes and peritoneal macrophages were cultured.Lymphocyte activation and specific cytotoxicity to tumor cells and toxicity of 2 ClA to macrophages were measured by MTT assay in the presence or absence of 2 ClA.Results:2 ClA had a strong cytotoxic effect on macrophages.When the activation test and cytotoxicity test of lymphocyte were measured by MTT assay,the optical density values of 2 ClA group was lower than that of control group,and statistic analysis showed P0 05.Conclusion:With the specific cytotoxicity to macrophages,2 ClA can exclude the influence of macrophages to MTT assay in activation test and cytotoxicity test of lymphocyte,and more reliable results will be obtained.
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Objective:Inhibition of alpha-glucosidase and antioxidation of different extracts from the branches and leaves of Taxus chinensis var mairei were studied in vitro to supply the separating base of the effective chemical components.Method:The inhibitory activities of α-glucoside were assayed in external.The antioxidant activities of these extracts were assessed bytwo complementary test systems,namely 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical scavenging activity and ferric reducing antioxidant power(FRAP) assay.Result:The ethyl acetate extract showed strong activity in the inhibitory activities of α-glucoside.(IC50 27.854 1 mg · L-1) The ethanol extract(IC50 29.215 5 mg · L-1)and n-butanol extract(IC50 38.913 0 mg · L-1) displayed lower activity.Petroleum ether fraction(IC50 128.729 5 mg · L-1) and water fraction(IC50 74.505 9 mg · L-1) have a little activity.The antioxidant activities:ethanol extract(DPPH IC50 6.370 7 mg · L-1,FRAP 4.266 mmol · g-1)ethyl acetate extract(DPPH IC50 9.535 8 mg · L-1,FRAP 3.275 mmol · g-1)n-butanol extract(DPPH IC50 20.461 3 mg · L-1,FRAP 1.498 mmol · g-1)water fraction(DPPH IC50 26.685 5 mg · L-1,FRAP 0.678 mmol · g-1)petroleum ether fraction.Conclusion: The components of inhibition α-glucoside could be testified in the ethyl acetate extract and n-butanol extract.Antioxidative components were in the ethyl acetate extract.
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Background: Medang lendir (Litsea glutinosa), a member of the Lauraceae family, is typically found in the Kampar Regency area of Riau Province, Indonesia. This plant is frequently used in traditional medicine to treat rheumatism, diarrhea, and dysentery. Objective: To evaluate the antioxidant activities of L. glutinosa stem bark extracts. Method: L. glutinosa stem bark was macerated in methanol to obtain methanol extract, followed by partitioning with ethyl acetate to obtain ethyl acetate extract. The antioxidant activities of these extracts were determined using the NO radical scavenging and the DPPH radical scavenging methods. The Folin-Ciocalteu method was also used in this study to determine total phenolic content (TPC). Result: The antioxidant activities of ethyl acetate and methanol extracts of L. glutinosa stem bark against NO radicals resulted in a small percentage of inhibition (%I < 0). Meanwhile, for DPPH radical scavenging, the antioxidant activity of the ethyl acetate extract (IC50 = 222.03 ug/mL) was greater than the methanol extract (IC50 = 232.81 ug/mL), and the TPC value of the ethyl acetate extract (23.88 mgGAE/g) was greater than the methanol extract (23.16 mgGAE/g). Conclusion: These results show that the antioxidant activity based on scavenging DPPH radicals for both extracts was active; however, it was inactive on NO radicals. The ethyl acetate extract possessed a higher TPC value compared to the methanol extract.
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Objective:To investigate 1,1-diphenyl-2-picrylhydrazy1(DPPH) free radical scavenging activities of extracts from Orobanche cernua var.cumana.Method:The 70% ethanol extracts from O.cernua var.cumana was extracted successively with ethyl acetate and n-butanol.The three fractions of ethyl acetate,n-butanol,and water were tested using assays of the scavenging effects on DPPH.The model of oxidative damage of human embryonic kidney cell line HEK-293 cells was induced by H2O2,MTT assay was used to evaluate the cell viability.Result:The IC50 in DPPH test were in the following orders:ethyl acetate(0.042 9 g · L-1)n-butanol(0.059 9 g · L-1)VC(0.071 8 g · L-1)water(0.330 3 g · L-1).The ethyl acetate fraction showed the strongest scavenging activity to DPPH free radicals.The three fractions of ethyl acetate,n-butanol and water could significantly improve the survival rate of injured HEK-293 cells,showing the antioxidant activities.Conclusion:These results demonstrate that the extracts from O.cernua var.cumana have some anti-oxidant and free radical scavenging activities.
Orobanche
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Background : Silver nanoparticles are of interest to be used as antimicrobial agents in medical care, wound dressings and cosmetics. Despite the fact that AgNPs are among the most commercialized nano materials owing to their specific antimicrobial properties, there is limited information about their risk assessment and possible hazards to human health and environment. Thus, this study was carried out to evaluate the cytotoxicity and morphological changes of different concentrations of two samples of commercialized silver nanoparticles (A and B) in CHO-K1 cells using MTT assay. Methods: The cytotoxicity effect of silver nanoparticles AgNP-A (19.6 nm in diameter) and AgNP-B (15 nm in diameter) on CHO-K1 was evaluated in the range of 0.005-500 µg/ml after 24 hours of treatment using MTT assay. The morphological changes of treated cells were examined by light microscopy. Results: Based on the results of cytotoxicity by MTT assay, reduced viability of cells and cytotoxicity were observed. The 50% inhibitory concentration (IC50) of silver nanoparticles was recorded at 100 and 10 µg/ml for AgNP-A and AgNP-B, respectively. Moreover, microscopic observations indicated clear morphological changes of treated cells. Conclusion: Concentration and physicochemical properties of silver nanoparticles like size, shape, surface area, coating and zeta potential play a role in cytotoxicity and morphological changes of treated cells.
Silver nanoparticle
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Zeta potential
Viability assay
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In this research work, a series of eighteen novel coumarinyl substituted thiazolidin-2,4-dione analogs (4a-4r) have been designed by molecular hybridization approach, synthesized and their structures were established on the basis of FTIR, 1H NMR, 13C NMR and elemental (CHN) analysis. These title compounds were screened for their cytotoxicity using MTT assay methodology against five different mammalian cancer cell lines viz. hormone dependant breast adenocarcinoma (MCF7), cervical carcinoma (HeLa), colorectal carcinoma (HT29), lung cancer (A549) and prostate adeno carcinoma (PC3). The cytotoxicity screening studies revealed that MCF-7, HeLa and A549 cancer cell lines were sensitive to all the tested compounds. Though the compounds showed varying degrees of cytotoxicity in the tested cell lines, most significant effect was observed for compounds 4i (1.06, 2.4 and 3.06 µM) and 4o (0.95, 3.2 and 2.38 μM) against MCF7, HeLa and A549 cell lines respectively. In conclusion, the anticancer results of these promising leads strongly encouraged us for additional lead optimization with the aim of developing more potential anticancer agents.
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MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.
MTT assay
Formazan
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Gambir (Uncaria gambir) and other plants belonging to the genus Uncaria have been used in traditional medicine in southeastern Asia, Africa and South America, and they have been studied widely over the past century. Gambier, the dried leaf extract from gambir is known to have antioxidant properties and some studies have attributed it to the presence of tannins and condensed tannins. The objective of this study was to investigate the potential of commercial gambier on the Indonesian market as a scavenger of reactive free radicals, evaluate its ability to inhibit α-glucosidase and determine the bioactive compound responsible for these activities. An ethanolic extract of commercial gambier was extracted with ethyl acetate. The ethanol and ethyl acetate extracts as well as the aqueous extract after ethyl acetate extraction and residue from ethanol extraction were tested for free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH). They were also tested for α-glucosidase inhibitory activity. The extracts were then studied using reverse phase HPLC, LCMS and NMR to identify the bioactive compound. It was observed that all the extracts had high activity for DPPH inhibition but moderate activity for inhibiting α-glucosidasein vitro. Apart from the aqueous extract, 92% DPPH inhibition by the extracts was achievable at 30 μg/ml. The ethanol and ethyl acetate extracts had significantly higher (p < 0.01) DPPH inhibitory activity than the aqueous extract.IC50 of the organic extracts and residue ranged between 13.8 to 16.2 μg/ml for DPPH inhibition while that of the aqueous extract was 27.4 μg/ml. With regards to α-glucosidase inhibition, however, IC50 range of 15.2 and 49.5 μg/ml was recorded. Catechin was identified as the major bioactive compound present.
Key words: Uncaria gambir, gambier, DPPH, alpha-glucosidase, HPLC, LCMS, NMR
Free radical scavenger
IC50
Residue (chemistry)
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