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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 70  |  Page : 396-403  

In vitro antitumor potential of methanol extract of Mimosa pudica in human breast cancer cell lines


1 Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala, India
2 Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Pookode, Wayanad, Kerala, India
3 Department of Veterinary Physiology, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala, India

Date of Submission26-Dec-2019
Date of Decision26-Feb-2020
Date of Acceptance21-Apr-2020
Date of Web Publication28-Aug-2020

Correspondence Address:
Bibu John Kariyil
Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Pookode - 673 576, Wayanad, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_527_19

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   Abstract 


Background: Mimosa pudica belonging to the family Fabaceae , is a small sized shrub, used traditionally for its anti-spasmodic, analgesic, anti-spasmodic, antibacterial, and antitumor activities. Objectives: The present study was conducted to assess the anticancer activity of methanol extract of whole plant of M. pudica (MMP) in MCF-7 and MDA-MB-231 breast cancer cell lines. Materials and Methods: The antitumor activity of MMP was analyzed using 3-(4,5-dimethyl thazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay in MCF-7 and MDA-MB-231 cell lines. The cytological and metabolic alterations due to MMP were evaluated using acridine orange/ethidium bromide dual staining, hoechst 33258 and fluoroprobe, benzimidazol-carbocyanine iodide 5, 5', 6, 6'-tetrachloro-1, 1', 3,3'-tetra ethyl (JC-1) staining. Real-time polymerase chain reaction and Western blotting were carried out to assess Bcl-2, the antiapoptotic gene, and protein expression, respectively. Qualitative analysis and gas chromatography high-resolution mass spectrometry were performed for the presence of various phytochemicals. Results: The methanol extract exhibited a potent antitumor activity in both the cell lines in vitro . Conclusion: The plant extract was showing the mode of action through intrinsic pathway of apoptotic cell death and may be studied further to develop a potent drug against breast cancer.

Keywords: 3-(4,5-dimethyl thazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, Bcl-2, gas chromatography high-resolution mass spectrometry, Mimosa pudica , phytochemical tests


How to cite this article:
John R, Kariyil BJ, A. Usha P T, Surya S, Anu G, John P, Sujith S, Zarina A. In vitro antitumor potential of methanol extract of Mimosa pudica in human breast cancer cell lines. Phcog Mag 2020;16:396-403

How to cite this URL:
John R, Kariyil BJ, A. Usha P T, Surya S, Anu G, John P, Sujith S, Zarina A. In vitro antitumor potential of methanol extract of Mimosa pudica in human breast cancer cell lines. Phcog Mag [serial online] 2020 [cited 2020 Oct 28];16:396-403. Available from: http://www.phcog.com/text.asp?2020/16/70/396/293780



SUMMARY

  • Phytochemical screening of the methanol extract of whole plant of Mimosa pudica revealed the presence of alkaloids, flavonoids, glycosides, steroids, phenolics, and diterpenes upon qualitative studies. Majority of compounds were belonging to terpenoids in the extract when assessed using gas chromatography high-resolution mass spectrometry. Since diterpenes were detected in phytochemical screening also, terpenoids might contributed for the obtained anticancer activity for the plant extract
  • On the basis of IC50 value calculated using 3-(4,5-dimethyl thazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, the methanol M. pudica extract possessed potent cytotoxic action. Methanol extract of the plant was found to induce apoptosis in breast carcinoma cells through intrinsic pathway which was assessed through acridine orange/ethidium bromide dual staining, hoechst staining, and JC-1 staining. The antiapoptotic gene and protein expressions of Bcl-2 were found to be significantly down regulated after the treatment with the plant extract. Further research and fractionation are required to characterize the bioactive compounds responsible for the obtained potential of M. pudica as a novel source for antitumor drugs.




Abbreviations used: MMP: Methanol extract of whole plant of Mimosa pudica ; MTT: 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; AO/EB: Acridine orange/ethidium bromide; JC-1: Benzimidiazol carbocyanine iodide 5,5', 6, 6'-tetrachloro-1,1', 3, 3'-tetra ethyl; GC-HRMS: Gas chromatography high-resolution mass spectrometry; NISCAIR: National Institute of Science Communication and Information Resources; CSIR: Council of Scientific and Industrial Research; NCCS: National Centre for Cell Sciences; ER: Estrogen receptor; PR: Progesterone receptor, HER: Human epidermal growth factor receptor; FBS: Foetal bovine serum; IC50: Half maximal inhibitory concentration; ANOVA: Analysis of variance; RT-qPCR: Real time-quantitative polymerase chain reaction; RIPA: Radioimmunoprecipitation assay; SDS-PAGE: Sodium dodecyl sulphate polyacrylamide gel electrophoresis; PVDF: Polyvinylidene difluoride; SAIF: Sophisticated Analytical Instrument Facility; IIT: Indian Institute of Technology; Δψm: Mitochondrial transmembrane potential; Bcl-2: B-cell lymphoma-2.


   Introduction Top


Herbal medicines have paved the way for the development of a huge number of drugs used for the cancer therapy nowadays. Recent research on cancer subtypes bring on evidence that medicinal plants and their derivatives could contribute to mainstream and supplementary therapeutic strategies in a tremendous manner. They are thought to augment therapeutic efficacy of conventional chemotherapy which may reduce their side effects in cancer patients.[1] Hence, the chemotherapeutic drug discovery field from herbal sources demand a sustained research to isolate newer compounds with maximum efficacy and limited toxicity at therapeutic levels.

The study mainly aims at the development of plant based novel drug molecules which could serve as a reliable alternative source for cancer chemotherapy. Mimosapudica belonging to the family Fabaceae , is a small-sized shrub, used traditionally for its anti-asthmatic, analgesic, anti-spasmodic, antibacterial, and antitumor activities.[2],[3] Taking into consideration on the traditional knowledge, this study focusses on the antitumor potential of methanol extract of M.pudica whole plant and an insight toward the probable phytoconstituents contributing to the outcome.


   Materials and Methods Top


Collection of plant material and authentication

The whole plants of M.pudica were collected during June–July 2016 from the Aranmula region of Pathanamthitta district, Kerala, India. The plant materials were authenticated by the Raw material and herbarium Department, National Institute of Science Communication and Information Resources (NISCAIR), Council of Scientific and Industrial Research and the voucher specimen of the plant was deposited in the Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Mannuthy with accession no. HERB/VPT/CVASMTY/2/2017.

Cell lines and culture conditions

Adherent human breast adenocarcinoma cell lines, MDA-MB-231 and MCF-7, procured from National Centre for Cell Science, Pune, Maharashtra, India, was utilized forin vitro anticancer studies. MDA-MB-231 cell line was estrogen receptor (ER) −ve, progesterone receptor (PR) −ve, and human epidermal growth factor receptor (HER) –ve. MCF-7 cell line was ER +ve, PR +ve, HER –ve. These adherent cells were grown in RPMI-1640 fortified with 10% foetal bovine serum and 1% antibiotic antimycotic solution (penicillin-streptomycin and amphotericin B), which were maintained in a humidified incubator at 37°C with 5% CO2. The cells were trypsinised using 0.25% trypsin/1 mM EDTA solution. Those cell suspensions having ≥95% viability (determined using trypan blue vital stain in automated cell counter (Countess, Invitrogen, Van Allen Way, Carlsbad, California) were used for seeding in culture plates for variousin vitro studies.

Methanol extraction

The whole plant of M.pudica was washed, shade-dried, and powdered coarsely in a temperature controlled plant sample pulverizer. The powdered plant material (1 kg) was extracted with methanol in soxhlet extractor at room temperature., The methanol extract, obtained after exhaustive extraction, was filtered and concentrated in rotary vacuum evaporator (Evator, Equitron EV11.ABI.029, India) at 50°C and 30 mmHg pressure. A reddish-brown colored residue obtained was first kept open at room temperature for complete evaporation of solvent and then stored in the sealed airtight container in refrigerator for further use.

Sample preparation

Stock solution of the methanol extract was prepared in 100% dimethyl sulfoxide (DMSO) at a concentration of 50 mg/mL. The filtered stock of extract was further diluted using phosphate buffered saline (PBS) to get the required concentrations of the extracts.

Cytotoxic evaluation of methanol extract of whole plant of Mimosapudicain vitro

In vitro cytotoxic potential of methanol extract of whole plant of Mimosapudica (MMP) was assessed in MCF-7 and MDA-MB-231 breast cancer cell lines, using 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) reduction assay as per Riss et al .[4] The cell concentration was maintained at 10000 cells per well, in 200 μL medium were incubated overnight at 37°C in CO2 incubator.

The extract stocks with concentrations ranging from 320, 160, 80, 40, 20, 10, 5, and 2.5 μg/mL were added to the cells for 48 h. After removing the extract added media, 20 μL of MTT (5 mg/mL prepared in Dulbecco's phosphate-buffered saline [DPBS]) was added and incubated at 37°C for 4 h in CO2 incubator. After incubation, the media-containing MTT was removed. Added 200 μL of DMSO (cell culture grade) to dissolve the formazan crystals formed. The absorbance was measured using microplate reader (Varioskan Flash, Thermofischer scientific, Finland) at a wavelength of 570 nm.

The percent cell viability was calculated using the following formula:

Percent cell viability = (average absorbance of treated cells/average absorbance of untreated cells) × 100.

The net absorbance from the wells of control cells was taken as 100% viable. IC50(half maximal inhibitory concentration) value of extract was calculated by plotting the concentration against percentage cell viability using the online software “very simple IC50 tool kit.”

Selection of concentrations

Three concentrations of the extract, i.e., one below IC50, IC50, and one above IC50 were selected for the study, based on the MTT assay. Thus the concentrations that were used for the study were 10, 20 and 40 and 8, 16 and 32 μg/mL for MDA-MB-231 and MCF-7 cells, respectively.

Microscopic studies

Trypsinized cells were seeded into 6 well cell culture plates at a cell concentration of 1 × 106 cells per well and incubated overnight. Cells were then treated with various concentrations of extracts for 24 h at 37°C and 5% CO2. Untreated cells were used as control. Doxorubicin at a concentration of 0.58 μg/mL was adopted as positive control. After 24 h of incubation the cells were trypsinised, washed with 1X DPBS and fixed with 4% paraformaldehyde in 1X PBS for 30 min at room temperature. The cells were again washed and resuspended in 50 μL DPBS. Acridine orange/ethidium bromide (AO/EB), hoechst 33258 and fluoroprobe, benzimidazol-carbocyanine iodide 5,5', 6, 6×-tetrachloro-1,1', 3, 3'-tetra ethyl (JC-1) staining techniques were used.

Dual acridine orange ethidium bromide staining

AO/EB staining was performed according to the method described by Kasibhatla et al .[5] in order to differentiate the live, apoptotic, and necrotic cells after treatment with the plant extract. Twenty-five microliters of cell suspension were mixed with 10 μL of AO/EB solution (1 part of l0 μg/mL acridine orange in PBS and 1 part of 10 μg/mL ethidium bromide in PBS) just before microscopy. Placed 10 μL of cell suspension on a microscopic slide, covered with cover slip and examined under trinocular Research Flourescent microscope (DM 200 LED Leica) with blue excitation (488 nm) and emission (550 nm) filters at 20×magnification.

Hoechst 33258 staining

Nuclear changes of apoptosis were determined by Hoechst 33258 staining.[6] MCF-7 and MDA-MB-231 cells (1 × 105 cells per well) were treated with the above-mentioned concentrations of MMP for 24 h. Cells were washed with PBS and fixed with methanol for 5 min. Cells were stained after fixation with Hoechsht 33258 stain in PBS (5 μg/mL) for 30 min at 37°C in the dark. Cells were thoroughly washed with PBS and examined under a fluorescence microscope (DM 200 LED Leica) with an excitation of 350 nm and emission of 460 nm filters at 200× magnification. The percent apoptotic cells were assessed by counting the number of apoptotic cells in six different microscopic fields. The variation between the groups for apoptotic cell percent were assessed by the one-way analysis of variance followed by Duncan's multiple comparison test.

JC-1 staining

MCF-7 and MDA-MB-231 cells were seeded in six well plates (1 × 105 cells/well) and treated with MMP in the above-mentioned concentrations for 24 h. 5 μM JC-1 stain was added and incubated at 37°C for 30 min in the dark. The cells were evaluated using Trinocular Research Fluorescence microscope (DM 2000 LED, Leica). The filters used were blue and red excitation/emission of 540/570 nm and 590/610 nm.[7]

In vitro Bcl-2 gene expression

The Bcl-2 gene expression in cell culture samples were evaluated using the real time-quantitative polymerase chain reaction (RT-qPCR). Corresponding IC50 concentrations of the extract were added to the cells for 24 h. The qRT-PCR was performed using Maxima SYBR green qPCR master mix (Thermo Scientific, USA) following the manufacturer's instructions. Reactions contained human Bcl-2 primer sets (Sigma). Human GAPDH served as a positive control. qRT-PCR was done on a Real time PCR cycler (Applied Biosystems, USA). The level of Bcl-2 expression was measured using the 2ΔΔCT method[8] and presented as fold change of the gene relative to the control cells. Expression fold change in gene and protein expression was assessed using the one sample t -test.

Western immunoblotting

Lysates of control and extract treated (IC50 concentration) cells were prepared by homogenizing cells with radio immunoprecipitation assay buffer with protease and phosphatase inhibitors on ice for 1 hr after washing twice in 1X PBS followed by centrifugation at 18,728 g, 4°C for 15 min. Total protein concentration was estimated by taking an aliquot of the lysate using Lowry method (Genei kit protocol). Using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, proteins were separated and subsequently transferred to polyvinylidene difluoride membrane (Hoefer Semi dry transfer apparatus). β actin was used as internal control to ensure equal protein loading. The membranes were incubated with primary antibodies of Bcl-2 (1:1000, Sigma-Aldrich) and beta-actin (1:2000, Sigma-Aldrich). The binding of antibodies were visualized by incubating the blots with horse radish peroxidase-conjugated secondary antibody (Cell Signaling Technology) followed by colour reaction with DAB substrate buffer. The Western blotting band strength was determined by Image J density Measurement program (http://imagej.en.softonic.com).[9] Expression fold change in protein expression was assessed using the one sample t -test.

Phytochemical screening

The methanol extract of M.pudica was subjected to preliminary phytochemical screening to study for the nature of various phytoconstituents.[10]

Gas chromatography high-resolution mass spectrometry analysis

Gas chromatography high-resolution mass spectrometry (GC-HRMS) analysis of MMP was conducted in Sophisticated Analytical Instrument Facility, Indian Institute of Technology, Mumbai, India. Gas chromatography (Agilent, USA, 7890) was used with a mass range of 10–2000 amu, mass resolution of 6000, FID detector, EI/CI source and time of flight analyser. The carrier gas used was helium at a flow rate of 1 ml/min. The oven temperature was increased to 200°C in 5 min after maintaining at 70°C for 1 min. The injector temperature was 250°C with a total analysis time of 50 min. After obtaining a clear baseline, 0.4 μL aliquots of extracts were injected into the chromatographic column. Interpretation on mass spectrum GC-MS and identification of major constituents were performed using mass spectrum library (NIST MS search 2.0 library).[11]


   Results Top


Cytotoxic evaluation of methanol extract of whole plant of Mimosapudicain vitro

In both MDA-MB-231 and MCF-7 cancer cell lines, the percent cell viability after addition of MMP showed an abrupt reduction followed by a static range from 20 μg/mL onward. The IC50 for MMP were obtained as 19.1 ± 8.28 and 16.07 ± 5.08 μg/mL for MDA-MB-231 cells and MCF-7 cells, respectively [Figure 1].
Figure 1: The percent cell viability of MDA-MB-231 and MCF-7 cells after 48 h treatment with methanol extract of Mimosa pudica determined by 3-(4,5-dimethyl thazol-2-yl)-2, 5-diphenyl tetrazolium bromide reduction assay. Values are expressed as mean ± standard error of the mean (n = 3)

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Dual acridine orange ethidium bromide staining

Live, necrotic, early and late apoptotic cells were detected after the treatment with extract. The representative images of cells of treatments after AO/EB staining are given in [Figure 2] and [Figure 3]. In control cell population, cells were live showing greenish fluorescence with circular nucleus uniformly distributed in the center. Early apoptotic cells with localized crescent-shaped or granular yellow-green stained nucleus were seen in both cell lines after the treatment with below IC50 concentrations of extract. Orange-to-red fluorescent cells in late apoptotic stage were seen with IC50 and above IC50 concentrations. Treated cells also showed obvious morphological changes such as membrane blebs, fragmentation of nuclei, chromatin condensation, and apoptotic bodies. Late apoptotic cells were seen mostly in doxorubicin-treated cells.
Figure 2: Morphological changes of MDA-MB-231 cells by acridine orange ethidium bromide staining, 200×. (a) Control cells; (b) Cells treated with doxorubicin 0.58 μg/mL; (c-e) Cells treated with methanol extract of Mimosa pudica at concentrations 10, 20, and 40 μg/mL, respectively. Green arrow-normal cells White arrow-early apoptotic cells, Yellow arrow-late apoptotic cells, Blue arrow indicates necrotised cells, Red arrow-nuclear fragmentation

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Figure 3: Morphological changes of MCF-7 cells by acridine orange ethidium bromide staining, 200×. (a) control cells; (b) Cells treated with doxorubicin 0.58 μg/mL; (c-e) cells treated with methanol extract of Mimosa pudica at concentrations 8, 16, and 32 μg/mL, respectively. Green arrow-normal cells, white arrow-early apoptotic cells, yellow arrow-late apoptotic cells, blue arrow indicates necrotised cells, red arrow-nuclear fragmentation

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Hoechst 33258 staining

In both control groups, uniform blue fluorescent live cells were obtained. Apoptotic characteristics, namely, nuclear fragmentation and marginalization, chromatin condensation were seen in extract treated and positive control cells [Figure 4] and [Figure 5]. Apoptotic cell percent showed a concentration-dependent significant increase (P < 0.01) [Figure 6].
Figure 4: Morphological changes of MDA-MB-231 cells by hoechst staining, 200×. A1-control cells; B1-Cells treated with doxorubicin 0.58 μg/mL; C1, D1, E1-cells treated with methanol extract of Mimosa pudica at concentrations 10, 20, and 40 μg/mL, respectively. White arrow – live cells, Red arrow-apoptotic cells, Yellow arrow-chromatin condensation, Green arrow-marginalisation of nucleus, Orange arrow-fragmentation of nuclei

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Figure 5: The apoptotic cell percent of MDA-MB-231 and MCF-7 cells after treatment with methanol extract of Mimosa pudica determined by Hoechst staining. Values are expressed as mean ± standard error of the mean (n = 6). Means with different superscripts differ significantly

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Figure 6: Morphological changes of MCF-7 cells by Hoechst staining, 200×. F1-control cells; G1-cells treated with doxorubicin 0.58 μg/mL; H1, I1, J1-cells treated with methanol extract of Mimosa pudica at concentrations 8, 16, and 32 μg/mL, respectively. White arrow - live cells, red arrow-apoptotic cells, yellow arrow-chromatin condensation, green arrow-marginalisation of nucleus, orange arrow fragmentation of nuclei

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JC-1 staining

In control cells, JC-1 aggregates with reddish/orange fluorescence were observed suggestive of a higher mitochondrial membrane potential. A dose dependent shift from red-to-green fluorescence was obtained for MMP after 24 h in both the cell lines, depicting a concentration dependent lowering of mitochondrial membrane potential [Figure 7] and [Figure 8].
Figure 7: Morphological changes of MDA-MB-231 cells studied by JC-1 staining, for methanol extract of whole plant of Mimosa pudica 200×. A2, C2, E2, G2 and I2-JC-1 Red; B2, D2, F2, H2 and J2-JC-1 Green A2, B2-control cells; C2, D2-Cells treated with doxorubicin 0.58 μg/mL; E2, F2, G2, H2, I2, J2-cells treated with methanol extract of Mimosa pudica at concentrations 10, 20 and 40 μg/mL, respectively

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Figure 8: Morphological changes of MCF-7 cells studied by JC-1 staining, 200×. K2, M2, O2, Q2 and S2-JC-1 Red; L2, N2, P2, R2 and T2-JC-1 Green K2, L2-control cells; M2, N2-Cells treated with doxorubicin 0.58 μg/mL; O2, P2, Q2, R2, S2, T2-cells treated with methanol extract of Mimosa pudica at concentrations 8, 16 and 32 μg/mL, respectively

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In vitro Bcl-2 gene expression

The relative Bcl-2 gene expression in the cell lines after MMP treatment is presented in [Figure 9]. The expression of Bcl-2 gene in the control cells was normalized to unity. In comparison to the control, Bcl2 gene expression after MMP treatment (IC50) decreased statistically significantly (P < 0.01) in both the cells with 0.88 ± 0.02 and 0.83 ± 0.02 folds for MDA-MB-231 and MCF-7 cells, respectively.
Figure 9: The relative Bcl-2 gene expression in MDA-MB-231 and MCF-7 cells in response to the treatment with methanol extract of Mimosa pudica. Values are expressed as mean ± standard error (n = 3) **denotes statistically significant (P < 0.01) difference compared with control

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Western immunoblotting

The treatment of MDA-MB-231 and MCF-7 cells with MMP (IC50 concentration) lowered the expression levels of anti-apoptotic protein Bcl-2 relatively [Figure 10]. Western blot images of β-actin and Bcl-2 proteins in MDA-MB-231 and MCF-7 cancer cells are presented in [Figure 11]A-D, respectively. A significant decrease in Bcl-2 protein expression (P < 0.01) with 0.89 ± 0.002 and 0.83 ± 0.012 fold change was observed for MDA-MB-231 and MCF-7 cells when compared with the control.
Figure 10: The relative Bcl-2 protein expression in MDA-MB-231 and MCF-7 cells in response to treatment with methanol extract of Mimosa pudica. Values are expressed as mean ± standard error (n = 3) **denotes significant (P < 0.01) difference compared with control

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Figure 11: A, B, C and D, Western blot images of β-actin and Bcl-2 proteins in MDA-MB-231 and MCF-7 cells. C and methanol extract of whole plant of Mimosa pudica are control cells and cells after treatment with methanol extract of whole plant of Mimosa pudica at its respective IC50concentration

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Phytochemical screening

The MMP shown the presence of alkaloids, flavonoids, glycosides, steroids, phenolics, and diterpenes upon analyzing phytochemical constituents using biochemical tests [Table 1].
Table 1: Phytochemical analysis of methanol extract of Mimosa pudica

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Gas chromatography high-resolution mass spectrometry analysis

The GC-HRMS analysis of MMP showed chromatogram with major peaks obtained at 18.66, 21.87, 37.49, 39.82, 40.27, 41.28, 43.52, and 44.10 min retention times [Figure 12]. The major phytochemicals identified using mass spectrum library were 2,4-bis (1,1-dimethyl ethyl) phenol, undecanoic acid, 2-hexadecen-1-ol, 3, 7, 11, 15-tetramethyl, carboxylic acid 4-oxazole, methyl, ethyl ester, 4,5 dihydro-2-phenyl-, 9, 12, octadecadienoyl chloride (Z, Z'), phytol, 17-octadecynoic acid, oleic acid, hexadecanoic acid, 3[(trimethyl silyl) oxy] propyl ester, oxirane tetradecyl, 9, 12, 15–octadecatrienoic acid, and Vitamin E which were mainly belonging to terpenoids, ether, fatty acid analogs, and sterols [Table 2].
Figure 12: Gas chromatography high-resolution mass spectrometry chromatogram of methanol extract of whole plant of Mimosa pudica

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Table 2: Gas chromatography . high.resolution mass spectrometry analysis of phytochemicals in the methanol extract of whole plant of Mimosa pudica

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   Discussion Top


Breast cancer, being one of the most frequently occurring types of carcinomas in humans, is responsible for the majority of carcinoma-related deaths. Oestrogen and progesterone play a pivotal lead in the treatment of certain breast cancers based on the presence of the corresponding receptors, owing the therapy to be classified to hormone responsive and nonresponsive types. Treatment regimens effective in both hormone-mediated and nonmediated types are of paramount importance as non-mediated types are difficult to diagnose, aggressive, and invasive in nature. Thus, the present study aimed at the development of novel agents which could be utilized in both these types of breast cancers.

Although there were few reports on the antiproliferative potential of M.pudica , a detailed study on the mode of action of the plant has not been made till date.[12],[13] MDA-MB-231 and MCF-7 cells were selected so that a compendious proposition on both hormone mediated and non-mediated carcinomas could be brought about from the current study.

With the use of MTT assay,in vitro antitumor activity of MMP was determined preliminarily in both the cell lines. The cells in active metabolism alone could reduce MTT into purple formazan product which has an absorbance maximum at 570 nm owing to the direct proportionality of colour change to cell viability.[4] As per NCI guidelines, the IC50 limit for selecting the plant extracts for anticancer studies is less than 30 μg/mL after 72 h of exposure.[14] Since IC50 value obtained in both the cell lines lie in this range, the extract could be selected and studied in detail as a source for a potential antitumor compound. Earlier studies suggested higher cytotoxic activity for methanol extract than hydroalcohol extract as the use of methanol as solvent derives more amount of potent cytotoxic phytoconstituents.[12] Mimosine, obtained from M.pudica inhibits DNA replication of breast cancer cells by targeting ribonucleotide reductase and serine hydroxy methyl transferase enzymes involved in dNTP synthesis with iron chelation in the initiation phase.[15]

MTT assay is incapable of detecting whether the cell growth inhibition has occurred due to apoptosis or necrosis. Drugs cause destruction of cancer cells mostly by inducing apoptosis whose sensitivity is directly proportional to the apoptotic levels.[16],[17] Dual AO/EB staining was used to assess apoptotic mode of cell death by morphological changes where a pronounced distinction between live, early, and late apoptotic cells and necrotic cells could be made. Cell penetration by AO stains the nuclei green by binding to DNA, especially normal and early apoptotic cells while EB stains the nucleus of late apoptotic and nerotic cells red whose plasma membrane integrity is lost by binding to DNA fragments and apoptotic bodies.[18],[19]

The effect on morphological changes in nucleus due to MMP in MDA-MB-231 and MCF-7 cells was assessed using hoechst 33258 staining. Hoechst-33258, a nuclear counter stain, emits blue fluorescence when bound to double-stranded DNA by causing intercalation between adenine and thymine residues. Apoptotic cells could be differentiated from viable cells by the emission of bright blue instead of uniform blue fluorescence. Chromatin condensation, nuclear marginalization, early nuclear collapse, and nucleosomal ladder formation are the major nuclear changes observed during apoptosis due to the involvement of caspases and other mitochondrial factors. Our results suggest that MMP is inducing apoptosis being evident from the nuclear changes with a significant dose-dependent increase in apoptotic cell percent.[20]

Apoptotic protein in intrinsic (mitochondrial dependent) pathway, targets mitochondria, increase mitochondrial membrane permeability causing leakage of apoptotic effectors by a fall in the mitochondrial transmembrane potential (Δψm). Due to high Δψm, JC-1 accumulates in mitochondrial matrix and form fluorescent red aggregates on lowering causes a decrease in red-to-green signal ratio, with increase in green monomer percent.[21] In the current study, MMP induced a decrease in Δψm in a dose-dependent manner by a mitochondrial-specific cationic dye, JC-1 showing probability for mitochondria dependent intrinsic pathway of apoptosis.[22],[23]

B-cell lymphoma-2 (Bcl-2) family proteins are significant in intermitochondrial membrane protein release. They could be classified into proapoptotic (Bax and Bak) and antiapoptotic (Bcl-2 and Bcl-xL) molecules. Bcl-2 suppresses apoptosis by blocking the release of cytochrome c from mitochondria, thereby inhibiting the subsequent activation of caspases associated with apoptotic cell death.[24],[25] A significant reduction in Bcl-2 gene and protein expression was obtained due to MMP in MDA-MB-231 and MCF-7 cells.[26],[27] The results represent the first report on the possible cellular mode of action of M.pudica extract on anticancer potential.

Alkaloids, flavonoids, glycosides, steroids, phenolics, and diterpenes were obtained on qualitative phytochemical analysis of MMP.[28] Previous studies suggested that flavonoids isolated from M.pudica present significant cytotoxic potential.[13] Phenolic acid exhibits marked antitumour and antimutagenic effect by hindering malignant tumor progression and flavonoids interfere in tumor development. Isoflavones often referred to as phytoestrogens, modulate estrogen levels, and thereby regulates hormone responsive tumor progression.[29]

GC-HRMS, a pivotal study for metabolic profiling, works on a combination of gas chromatography and mass spectrum division patterns with a database for detecting phytochemicals. Nowadays, GC-MS evolved as a key method for metabolic profiling.

Among the major compounds obtained with GC-HRMS, there are early records on the detection of 9, 12, octadecadienoyl chloride (Z, Z'), phytol, 17-octadecynoic acid, hexadecanoic acid, and Vitamin E.[28],[30],[31] Phytol possess anticancer activity and act as precursor for Vitamin E, a well-known antioxidant.[32] Potent antitumor activity by terpenoids has also been reported.[33]


   Conclusion Top


Thus, the present study revealed that MMP possess antitumour activityin vitro against both MDA-MB-231 and MCF-7 cell lines. The extract was able to produce considerable cytotoxicity against these cell lines causing apoptosis of the cancer cells through intrinsic pathway as evidenced by AO/EB, Hoechst 33258, and JC-1 staining. The extract was causing a downregulation of the antiapoptotic gene and protein Bcl-2, which substantiate the potential to induce apoptosis in vitro . Terpenoids obtained on both phytochemical and GC-HRMS analysis may be a contributing factor for the obtained anticancer activity in vitro .

Acknowledgements

The authors are thankful to Sophisticated Analytical Instrumental Facility, Indian Institute of Technology, Mumbai, India, for help in conducting GC-HRMS analysis.

Financial support and sponsorship

The authors acknowledge the financial supports provided by Government of Kerala as State Plan project titled “Screening and evaluation of medicinal plants for anticancer activity” with grant No. RSP/16-17/VII-6 and Kerala Veterinary and Animal Sciences University research grant no. AD/2/96/MVM/2015/PH.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
 
 
    Tables

  [Table 1], [Table 2]



 

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