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ORIGINAL ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 1  |  Page : 7-10  

In vitro cytotoxicity evaluation of stem bark and leaves of Anogeissus pendula: Lack of potent activity


1 Department of Pharmacy, Banasthali University, Banasthali, Rajasthan; Department of Pharmacology, Khalsa College of Pharmacy, Amritsar, Punjab, India
2 Department of Pharmaceutical Chemistry and Analysis, Khalsa College of Pharmacy, Amritsar, Punjab, India
3 Department of Pharmacy, Banasthali University, Banasthali, Rajasthan, India

Date of Web Publication21-Apr-2017

Correspondence Address:
Rakesh Yadav
Department of Pharmacy, Banasthali University, P.O. Banasthali University, Tonk, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-9234.204915

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   Abstract 

Objective: Anogeissus pendula Edgew (Combretaceae) which is also known as “button tree” is one of the prominent species of Anogeissus genus which has several ethnomedicinal uses. Phytochemical investigation revealed the presence of alkaloids and phenolic compounds, such as flavonoids, lignins, tannins. So, pharmacological evaluation of this plant may lead to discovery of new activity. Therefore, the objective of the present study is to evaluate the in vitro cytotoxicity of stem bark and leaves hydroalcoholic extract of A. pendula. Materials and Methods: In vitro cytotoxicity was evaluated by MTT (3-(4,5-dimethyl thiazol-2-yl)-5-diphenyl tetrazolium bromide) assay against A431 (human epithelial carcinoma) and HepG-2 (human liver carcinoma) cell lines. The assay is based on the principle that the dead cells do not reduce the tetrazolium into formazan. Various concentrations (62.5-1000 μg/mL) of hydroalcoholic extracts were used and the concentration of test drug needed to inhibit cell growth by 50% (IC50) values is generated from the dose–response curves for each cell line. Microscopic examination was also carried out for morphological analysis of cells treated with extracts of A. pendula. Results: The hydroalcoholic extracts of stem bark and leaves revealed dose dependent, but insignificant activity against the cell lines i.e.A431 and HepG-2 as the IC50 was found to be greater than 1000 μg/mL for both the extracts against both the cell lines. There were no morphological changes in cells which supports the insignificant cytotoxicity. Conclusion: The results suggest that the hydroalcoholic extracts of stem bark and leaves of A. pendula have insignificant cytotoxicity.

Keywords: Anogeissus pendula, cytotoxicity, hydroalcoholic extracts, in vitro, MTT assay


How to cite this article:
Singh D, Baghel U S, Yadav R. In vitro cytotoxicity evaluation of stem bark and leaves of Anogeissus pendula: Lack of potent activity. J Pharm Negative Results 2017;8:7-10

How to cite this URL:
Singh D, Baghel U S, Yadav R. In vitro cytotoxicity evaluation of stem bark and leaves of Anogeissus pendula: Lack of potent activity. J Pharm Negative Results [serial online] 2017 [cited 2017 May 26];8:7-10. Available from: http://www.pnrjournal.com/text.asp?2017/8/1/7/204915


   Introduction Top


Cancer is a major cause of mortality worldwide commonly due to the lack of widespread and comprehensive early detection methods and the related poor prognosis of patients diagnosed in later stages of the disease and also due to increasing incidences. Major etiological factors involve the unhealthy lifestyle and pollution.[1],[2] Despite of amazing developments in modern medicine, such as radiotherapy, chemotherapy, hormone therapy, and surgery, the search for new alternative is still under investigation due to the enormous side effects of the mentioned therapies. There is considerable scientific and commercial interest in the discovery of new anticancer agents from natural product sources and it is evident that the nature has always been a great contributor.[3] Chemoprevention is accepted as an important approach to control malignancy and the search for desirable chemopreventive agents is the main area of focus for recent studies. The involvement of free radicals in carcinogenesis is well documented as they could aid in cancer development by DNA or other biomolecules damaging. Natural products, particularly dietary substances containing antioxidant properties, have played an important role in creating new chemopreventive agents as they possess ability to inhibit carcinogenesis.[2],[4] Several natural products of plant origin have potential value as chemotherapeutic agents, such as vinca alkaloids (vincristine, vinblastine and vindesine) and the podophylotoxin derivatives (etoposide and teniposide).[5] These facts support the evaluation of local medicinal plants for anticancer properties.

Anogeissus pendula Edgew (Combretaceae) also known as “button tree”.[6] It is a gregarious shrub or small tree and grows in dry and mixed forests of India.[7],[8] It has various ethnomedicinal uses such as in gastric disorder, wound healing, skin diseases, diarrhoea, dysentery, cough and burns of stem bark, seed, fruit, and twig.[9],[10],[11],[12] Phytochemical investigation revealed the presence of alkaloids and phenolic compounds, such as flavonoids, lignins, and tannins.[13],[14],[15] On the basis of the ethnomedicinal uses in skin diseases and gastric disorders along with the presence of phenolic compounds, the plant was evaluated for cytotoxicity for the first time.


   Materials and Methods Top


Plant Material and Extraction

The plant parts were collected from Sawai Madhopur (Rajasthan) in the month of November. The plant was identified and authenticated by Dr. Krishnendra Singh Nama, Botanist, Maa Bharti P.G. College, Kota. Collected plant parts were shade dried and powdered coarsely. Standard procedure and analytical grade solvents were used for the extraction. 40 g of plant parts' powder (stem bark and leaf) was preliminary extracted with chloroform and n-hexane, respectively by Soxhlet apparatus and further with the same procedure extracted with 60% ethanol. Obtained extracts were concentrated under reduced pressure and freeze-dried to get yield of 9.2% (stem bark; APB) and 20.1% (leaf; APL). Extracts were stored in sealed vials in a freezer until tested.

Chemicals

3-(4,5-dimethyl thiazol-2-yl)-5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), phosphate buffered saline (PBS), minimum essential medium (MEM), Dulbecco's modified eagle medium (DMEM), trypsin (Sigma Aldrich); ethylenediaminetetraacetic acid (EDTA), glucose, antibiotics (penicillin, streptomycin, amphotericin B) (Hi-Media); dimethyl sulfoxide, propanol (E. Merck).

Cell Lines

431A (human epithelial carcinoma) and HepG-2 (human liver carcinoma) cell lines were procured from National Centre for Cell Sciences, Pune, India. Stock cells were cultured in MEM and DMEM respectively, supplemented with 10% inactivated FBS, penicillin (100 IU/mL), streptomycin (100 μg/mL), and amphotericin B (5 μg/mL) in a humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with TPVG (0.2% trypsin, 0.02% ethylenediaminetetraacetic acid (EDTA), 0.05% glucose in PBS) solution. The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 microtiter plates (Tarsons India Pvt. Ltd., Kolkata, India).

Cytotoxicity Assay

The assay was carried out according to Francis and Rita (1986). The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 105 cells/mL using MEM and DMEM containing 10% FBS. To each well of the 96 well microtiter plate, 0.1 mL of the diluted cell suspension (approximately 10,000 cells) was added. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 μL of different test concentrations (62.5-1000 μg/mL) of test drugs were added on to the partial monolayer in microtiter plates. The plates were then incubated at 37°C for 3 days in 5% CO2 atmosphere. Microscopic examination was carried out and observations were noted every 24 hours interval for morphological analysis of cells treated with extracts of A. pendula. After 72 hours, the drug solutions in the wells were discarded and 50 μL of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 hours at 37° C in 5% CO2 atmosphere. The supernatant was removed and 100 μL of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC50) values is generated from the dose–response curves for each cell line.




   Results Top


Proliferative Effect

The anticancer effect of extracts of A. pendula on A431 and HepG-2 cell lines was evaluated through microculture MTT assay. The multiple concentrations (62.5-1000 μg/mL) of hydroalcoholic extracts were used and the concentration needed to inhibit cell growth by 50% (IC50) value is generated from the dose-response curves for each cell line. Results of the cytotoxicity evaluation against A431 and HepG-2 cell lines of the A. Pendula extracts are shown in [Figures 1] and [Figures 2]. Both the hydroalcoholic extracts of stem bark and leaves revealed dose dependent but insignificant activity against both the cell lines with IC50 greater than 1000 μg/mL. The insignificant cytotoxicity was also supported by the microscopic evaluation for morphological changes in cells but there are none.
Figure 1: Cytotoxic effect of APB and APL against A431 (Human epithelial carcinoma) cell line.(at column width)

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Figure 2: Cytotoxic effect of APB and APL against HepG2 (human liver carcinoma) cell line.(at column width)

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   Discussion Top


Plants hold almost unlimited capability to produce bioactive compounds that attracts investigators in the pursuit of novel chemotherapeutic agents. The search of new chemotherapeutics in plants and dietary components is a promising approach.[17] Our recent study reported the in vitro cytotoxicity evaluation of hydroalcoholic extracts of stem bark and leaves of A. pendula by MTT assay. The ability of the cells to survive a toxic insult has been the basis of most cytotoxicity assays. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the number of cells present and on the mitochondrial activity per cell. The principle involved is the cleavage of tetrazolium salt that is MTT into a blue colored product (formazan) by mitochondrial enzyme succinate dehydrogenase. The number of cells was found to be proportional to the extent of formazan production by the cells. A. pendula was chosen for this study due to the various ethnomedicinal uses like in skin diseases and gastric disorders.

Literature survey supports the presence of phenolic compounds in the plant.[13],[14],[15] Phenolic compounds are responsible for the antioxidation and antiproliferation. Natural antioxidants inhibit tumor growth selectively due to the difference in redox status of normal cell and cancer cell.[18] In the present study, both the extracts of stem bark and leaves of A. pendula was found to had insignificant cytotoxicity against A431 (human epithelial carcinoma) and HepG-2 (human liver carcinoma) cell lines with IC50 greater than 1000 μg/mL. According to U.S. National Cancer Institute (NCI) guidelines the limit of activity for crude extracts is IC50 less than 30 μg/mL after 72 hours of exposure. However, a crude extract with IC50 less than 20 μg/mL is considered highly cytotoxic.[17] The IC50 of the plant under study was found to be above which is specified by NCI, USA for categorization of compound as chemotherapeutic. Therefore, stem bark and leaves of A. pendula has insignificant cytotoxic potential against A431 and HepG2 cell lines. This study will be helpful to avoid any repeated study in this direction in the future.


   Conclusion Top


The hydroalcoholic extracts of stem bark and leaves of A. pendula had insignificant cytotoxic potential against A431 and HepG2 cell lines which was evaluated by MTT assay.

Acknowledgement

Authors are thankful to the Vice-chancellor, Banasthali University for providing necessary research facilities. Thanks are also due to DST, India for providing DST-CURIE grant to the university.

Financial support and sponsorship

Nil

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Kinghorn AD, Farnsworth NR, Soejarto DD, Cordell GA, Swanson SM, Pezzuto JM, et al. Novel strategies for the discovery of plant derived anticancer agents. Pharmaceutic Biol 2003;41:53-67.  Back to cited text no. 3
    
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Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nature Rev Cancer 2003;3:768-80.  Back to cited text no. 4
    
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Scott AJ. A revision of Combretaceae. Kew Bull 1979;33:555-66.  Back to cited text no. 8
    
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Jain A, Katewa SS, Galav PK, Sharma P. Medicinal plant diversity of Sitamata wildlife sanctuary, Rajasthan, India. J Ethnopharmacol 2005;102:143-57.  Back to cited text no. 9
    
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Ray S, Sheikh M, Mishra S. Ethnomedicinal plants used by tribals of east Nimar region, Madhya Pradesh. Indian J Tradit Know 2011;10:367-71.  Back to cited text no. 10
    
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Sikarwar RLS, Pathak B, Jaiswal A. Some unique ethnomedicinal perceptions of tribal communities of Chitrakoot, Madhya Pradesh. Indian J Tradit Know 2008;7:613-7.  Back to cited text no. 11
    
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Mishra A. Study on some ethnomedicinal plants of Kalinjar hillock, Banda district (U.P) India. Int J Adv Res Eng Appl Sci 2015;4:1-9.  Back to cited text no. 12
    
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Hungund BL, Pathak CH. A survey of plants in Gujarat, India for alkaloids, saponins, and tannins. U.S.D.A. Forest Service Research Paper 1971;NE-201:1-11.  Back to cited text no. 13
    
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Ramana DBV, Singh S, Solanki KR, Negi AS. Nutritive evaluation of some nitrogen and non-nitrogen fixing multipurpose tree species. Anim Feed Sci Technol 2000;88:103-11.  Back to cited text no. 14
    
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Arunadevi R, Lata S, Bhadoria BK, Ramteke VD, Kumar S, Sankar P. Neuroprotective effect of 5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β-D-glucopyranoside, a polyphenolic compound in focal cerebral ischemia in rat. Eur J Pharmacol 2010;626:205-12.  Back to cited text no. 15
    
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Francis D, Rita L. Rapid colorometric assay for cell growth and survival modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986;89:271-7.  Back to cited text no. 16
    
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Vijayarathna S, Sasidharan S. Cytotoxiciy of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines. Asian Pac J Trop Biomed 2012;2:826-9.  Back to cited text no. 17
    
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    Figures

  [Figure 1], [Figure 2]



 

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