{"id":868,"date":"2015-02-16T08:00:29","date_gmt":"2015-02-16T08:00:29","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=868"},"modified":"2020-04-25T08:17:56","modified_gmt":"2020-04-25T08:17:56","slug":"study-on-the-apoptotic-properties-of-methanolic-extracts-of-peltophorum-pterocarpum-cassia-auriculata-cassia-alata-and-lamprachaenium-microcephalum","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol2no2\/study-on-the-apoptotic-properties-of-methanolic-extracts-of-peltophorum-pterocarpum-cassia-auriculata-cassia-alata-and-lamprachaenium-microcephalum\/","title":{"rendered":"Study on the Apoptotic Properties of Methanolic Extracts of Peltophorum Pterocarpum, Cassia Auriculata, Cassia Alata and Lamprachaenium Microcephalum"},"content":{"rendered":"

Introduction <\/strong><\/p>\n

Cancer is one of most dreadful diseases of 20th\u00a0and 21st\u00a0century. Although people of all ages develop cancer, most types are more common in people over the age of 50. Cancer usually develops gradually over many years, the result of a complex mix of environmental, nutritional, behavioral, and hereditary factors. Apoptosis is a fundamental physiological process in mammals in which cells die by activating an Intrinsic Suicide Mechanism1. Defects in apoptotic signaling pathways play critical roles in a multiplicity of pathophysiological status including cancer. The highly regulated systematic nature of apoptosis lends itself to distinct morphological and biochemical criteria including selective and tightly controlled activation of proteolytic cascades that result in an ordered disassembly of cells2.<\/p>\n

Modern surgery had significantly reduced the cancer mortality rate, chemotherapy and radiation therapy were reducing the death rate not more than 5% and they are producing numerous side effects. But majority of tumor cells were resistant to these therapies therefore search for the alternative agent which can cure tumor without any side effects is under way. Among them, herbal plants are used as complementary and alternative medicine. Use of herbal medicines in Asia represents a long history of human interactions with the environment.\u00a0 According to World Health organization (WHO) more than 80% of the World\u2019s population relies on traditional medicine for their primary health care. These medicinal herbs were used for long time to cure illness and some of these plants were believed to promote resistance against infection. Recently great emphasize is given towards the complementary and alternative medicine treatment. Ayurveda is one of the best complementary medicinal treatments for cancer therapy. Ayurveda describes cancer as inflammatory or non-inflammatory swellings3.<\/p>\n

Herbal decoctions consisting of multiple herbs each possessing tremendous potential for a cancer cure are commonly used in Ayurveda. These formulations are reported to work on multiple biochemical pathways and are capable of influencing several organ systems simultaneously. The benefit of a herbal decoction is that it can nourish the body as a whole by supporting various organ systems.<\/p>\n

In the present study, methanolic extracts of the leaves of Peltophorum pterocarpum<\/em>, Cassia auriculata<\/em>, Cassia alata<\/em> and Lamprachaenium microcephalum<\/em>\u00a0 have been tested for the ability to induce cell death in cancer cell line by apoptosis as determined by AOEB \/ HOESCHT 33258 staining and morphological examination.<\/p>\n

Material and Methods<\/strong><\/p>\n

Plant Collection and Methanolic Extraction<\/strong><\/p>\n

Plants such as Cassia auriculata,<\/em> Cassia alata, Peltophorum pterocarpum and Lamprachaenium microcephalum<\/em> were collected from places around Tiruchirappalli District and identified by the Postgraduate and Research Department of Plant Biology and Plant Biotechnology, St. Joseph\u2019s college (Autonomous), Tiruchirappalli. Leaves of the plants were dried under shade, powdered and extracted using methanol as solvent by hot extraction method with Soxhlet apparatus. The extracts were collected, dried and used for treating the cells at various concentrations4.<\/p>\n

Cell lines<\/strong><\/p>\n

MCF-7 Cell line was derived from National Centre for Cell Science, Pune.\u00a0 The Cell line was cultured in RPMI- C medium with 10% Foetal Bovine Serum.\u00a0 The cell line was cultured at pH 7.1, temperature 370C with 5% CO2. All reagents and media were purchased from Himedia, Mumbai, India.<\/p>\n

RPMI Complete Medium (RPMI-C)<\/strong><\/p>\n

RPMI \u2013 1640 was supplemented with 10% heat \u2013 inactivated foetal Bovine serum (FBS), 2 mM L- Glutamine, 20mM HEPES Buffer, 100\u00b5g\/ml penicillin, 150\u00b5g\/ml streptomycin and 50\u00b5M 2-mercapto-ethanol.<\/p>\n

MTT Assay<\/strong><\/p>\n

MTT (3 – 4,5 – dimethyl thiazol-2-yl) 2-5, diphenyl tetrazolium bromide), a pale yellow substrate is converted into a formazan, a violet compound by the activity of succinate dehydrogenase of mitochondria. Since the conversion takes place in living cells, the amount of formazan is directly correlated with the number of viable cells present. MTT assay was done following the method of Mosmann with slight modification5,6,7. In brief, cells (1\u00d7105\u00a0per well in 200\u00b5l medium) were seeded in 96 well plate and allowed to adhere for 24 hours at 370C with 5% CO2\u00a0in air. Medium was aspirated and replaced with medium containing methanolic extracts in DMSO in concentrations ranging from 1, 10, 100, 1000 and 10000mg\/ml for 24 hours at 370C with 5% CO2\u00a0in air. Then 10\u00b5l of MTT (5mg\/ml stock solution) in PBS was added to each well containing 100 \u00b5l of cell suspension and re-incubated for 4 h at 370C. The reaction mixture was carefully taken out and 200 \u00b5l of DMSO was added to each well and mixed thoroughly. After 10 minutes, the color was read at 530nm using multi-well microplate reader (BIORAD, USA). The untreated sets were also run parallel under the identical conditions and served as \u2018Control\u2019.<\/p>\n

AOEB Staining<\/strong><\/p>\n

MCF-7 cells were cultured in RPMI-C at 37\u00b0C in a humidified atmosphere of 5%CO2 in air for 24 or 48 h in the presence or absence of each methanolic extracts of the leaves mentioned above. After treatment, 100\u00b5l of these cell suspensions (1\u00d7105\u00a0cells\/ml) in RPMI-1640 were collected, centrifuged at 200g for 1 minute and mixed with 1\u00b5l of dye mix (1\u00b5g\/ml acridine orange plus 1\u00b5g\/ml Ethidium bromide in PBS). Observation was carried out at 400x using an epi-illumination microscope (Olympus, Japan) with a filter combination suitable for fluorescein visualization. At least 200 cells were counted and the number of cells with fragmented nuclei, increased cytoplasm and condensed chromatin, which reliably indicate apoptosis, were determined as previously described8,9,10.<\/p>\n

Hoechst H-33258 Staining<\/strong><\/p>\n

About 1\u00d7105\u00a0cells were washed twice with PBS and then re-suspended in 500 \u00b5l of Ice cold methanol and placed at 40C for 5 minutes. Cells were washed once with PBS and resuspended in a solution of Hoescht H-33258 nuclear stain (Sigma) (1\/100 dilution of Hoescht H-33258 in PBS) and placed for 5 minutes at 40C (Light protected). Samples were subsequently washed twice with PBS and finally the pellet was resuspended 20\u00b5l of PBS. Cells were spread on microscope mounting plate, covered with a cover slip and analyzed under a Fluorescence Microscope (Olympus, Japan) with emission at 492 nm with excitation at 356 nm. Nuclei exhibiting fragmentation and disintegration were observed.<\/p>\n

Results and Discussion<\/strong><\/p>\n

Cell Viability Assay<\/strong><\/p>\n

As determined by MTT assay, the methanolic extracts of the leaves of Peltophorum pterocarpum<\/em>, Cassia auriculata<\/em>, Cassia alata<\/em> and Lamprachaenium microcephalum<\/em> strongly affected the survival of the MCF-7 cell line in time and dose dependent manner. MCF-7 cell line is a breast cancer cell line being very sensitive to cytolytic agents and is easily maintained. In the study, the cells were treated with various concentrations of the extracts ranging from 1, 10, 100, 1000 and 10,000 mg\/ml and the cell viability was measured by MTT assay. The inhibition of the cell viability was clearly observed in a dose \u2013dependent manner. The IC50\u00a0value was 3mg\/ml for Peltophorum pterocarpum<\/em> and Cassia auriculata<\/em> and 1mg\/ml for Lamprachaenium microcephalum<\/em> and Cassia alata<\/em> for MCR-7 after 24 hours (Table 1).<\/p>\n\n\n\n
\"Table<\/td>\nTable 1: IC50\u00a0of methanolic extracts at 24 and 48 Hours.<\/strong><\/p>\n

 <\/p>\n

Click here to View Table<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Analysis of Apoptosis<\/strong><\/p>\n

Acridine orange (AO) – Ethidium bromide (EB) and Hoechst staining were performed to evidence possible changes in cellular morphology. The control cells appeared green and blue respectively (Fig.1and 2), whereas methanolic extract – treated cells were shown to possess abnormalities. Microscopic images of the cells treated with Lamprachaenium microcephalum<\/em> and Cassia alata<\/em> revealed nuclear alterations typical of the apoptotic process. Both the stains revealed cytoplasmic blebbling, presence of apoptotic bodies, marginalization of chromatin and innumerable micronuclei in cells treated with both the methanolic extracts of Lamprachaenium microcephalum<\/em> and Cassia alata <\/em>(Fig 1and 2).. On the other hand, death pattern of cells treated with Cassia auriculata<\/em> and Peltophorum pterocarpum<\/em> was much similar to necrosis.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1: Microscopic views of MCF – 7 cell lines treated with Lamprachaenium microphalum.<\/em><\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\nFigure 2: Microscopic views of MCF – 7 cell lines treated with Cassia alata.<\/em><\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Conclusion<\/strong><\/p>\n

Results from this study allow us to demonstrate that it is mainly the extracts of Lamprachaenium microcephalum<\/em> and Cassia alata<\/em> to inhibit tumor cells. Such inhibition may be mediated by Apoptosis. It is also further implicated from the study that the methanolic extracts of Lamprachaenium microcephalum<\/em> and Cassia alata<\/em> can be re-extracted with other solvent such as Chloroform, ethyl acetate and water for further analysis into the suspected apoptosis mediated cell death.<\/p>\n

Acknowledgements<\/strong><\/p>\n

The authors are thankful to Dr. M. Sheik Mohamed, Principal, Dr. M. M. Shahul Hameed, Head, Dept. of Biotechnology, and the Management, Jamal Mohamed College (Autonomous), Tiruchirappalli \u2013 620 020, Tamil Nadu, for their constant support and provision of the facilities.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Kerr, J. F. \u201cHistory of the events leading to the formulation of the apoptosis concept.\u201d Toxicology,<\/em> 181-182 (2002).<\/li>\n
  2. Green, D.R. and Evam, G. L. \u201cA matter of life and death.\u201d Cancer Cell,<\/em> 19 \u2013 30 (2002).<\/li>\n
  3. Rama Ranga, S. \u201cA herbal medicine for the treatment of lung cancer.\u201d Molecular and Cellular Biochemistry,<\/em> 280: <\/strong>125-133 (2005).<\/li>\n
  4. Premalatha Balachandran and Rajagopal Govindarajan. \u201cCancer an ayurvedic prespective.\u201d Pharmacological Research,<\/em> 51:<\/strong> 19-30 (2005).<\/li>\n
  5. Mosmann, T. \u201cRapid colorimetric assay for the growth and survival: Application to Proliferation and Cytotoxicity assays.\u201d Journal of Immunolological methods, <\/em>65<\/strong>: 55-63(1983).<\/li>\n
  6. Raj Upreti, K., Kannan, A. and Pant, A. B. \u201cExperimental exposure of Arsenic in cultured at intestinal epithelial cells and cell line: Toxicological consequences.\u201d Toxicology in Vitro,<\/em> 21<\/strong>: 32-40 (2007).<\/li>\n
  7. Mosmann. \u201cMTT assay.\u201d J. Immunol. Methods,<\/em> 65:<\/strong> 55 (1983).<\/li>\n
  8. Caldus Lopes, E., Garcaa, M.G., Vallon, L., Alvarez, E. and Hajos, S.E. \u201cCorrelation with decreased apoptosis and multi \u2013 drug resistance in murine leukemic T Cell lines.\u201d Leuk. Lymphoma,<\/em> 42<\/strong>: 775-787(2001).<\/li>\n
  9. Kurz, E.U., Wilson, S.E., Leader, K.B., Sampey, B.P., Allan, W.P., Yalowich, J.C. and Kroll, D.J. \u201cThe histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to Etoposide in human leukemic cell lines.\u201d Mol.Cancer Ther,<\/em> 1<\/strong>: 121-131(2001).<\/li>\n
  10. O\u2019Connell, A.R., Holohan, C., Torriglia, A., Lee, B.F. and Stenson-Cox, C. \u201cCharacterization of a serine protease-mediated cell death program activated in human leukemia cells.\u201d Experimental Cell Research,<\/em> 312<\/strong>: 27-39 (2006).<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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