Enhancement of Anticancer Drug Annona muricata Against HT-29 Cell Line using Silver Nano Particles

 

Arvindganth. R, Anupriya K.V., Kathiravan. G*

Vels University, Pallavaram, Chennai.

 

ABSTRACT:

This study reports in vitro cytotoxicity effect of biogenic synthesized silver nanoparticles against HT-29 colon cancer cell line. The formation of silver nanoparticles was observed at 449nm, further the various characterization techniques such as UV, SEM, IR studies were conformed the synthesis of silver nanoparticles. The plant extract derived nanoparticle sample was exhibited significant cytotoxicity effects against HT-29 colon cancer cell line. Thus, the results shows the present study indicates the biogenic synthesized silver nanoparticles might be used to treat colon cancer cell line and it’s followed to their potential as anticancer agents.

 

 

INTRODUCTION:

All over the world the herbal medicine acts as the representative of the most important fields of traditional medicine. The study on the medicinal plants is essential to promote the proper use of herbal medicine in order to determine their potential as a source for the new drugs. [1]. Plants and plant-based medicaments are used as the basis of many of the modern pharmaceuticals that we use today in order to treat our various ailments. The better understanding of the plant derived medicine depends mainly on two factors that have gone hand in hand. [2]. One criterion involves the proof to show that the formulated medicine does what it is claimed to do and other is the identification of the active compound by means of the chemical analysis. The term "Annonaceae" refers to plants in the Annonaceae family, which is a member of the order Magnoliales. It is the largest of the families in that order, with more than 120 genera and more than 2,200 species. [3]. the common name of the family is custard apple family.

 

Most plants in this family are shrubs and small trees, and a few species are woody vines of the type lianas. The family has both deciduous and evergreen trees. The common characteristic of this family includes aromatic bark and having two rows of smooth-edged leaves arranged alternately on their branches. Flowers of this family usually have shades of yellow or white and have six petals and multiple pistils. Most plants in this family also have the sepals at the base of their flowers divided into three sections. [4].The fruits, which develop from the pistils, may be borne singly or in clusters. In some species the developing ovaries merge into multi-part fruits. The Annonaceae family can be found worldwide. Most of them are tropical or sub-tropical and a wide variety are found in the jungles of the Amazon. Fruit-bearing species primarily originated in the America, but are has spread to all tropical regions of the world by humans. Species of Annonaceae were widespread outside the tropics, but the majority of non-tropical genera and species have died out. [5]. some species, such as the custard apple itself, can be grown as far north as Florida. The pawpaw tree of the American southeast, Asimina triloba, is a surviving temperate-zone representative of the family. The methods for making nanoparticles can generally involve either a “top down” approach or a “bottom up” approach [6] In top-down synthesisn, nanoparticles are produced by size reduction from a suitable starting material [7].Size reduction is achieved by various physical and chemical treatments. Top down production methods introduce imperfections in the surface structure of the product and this is a major limitation because the surface chemistry and the other physical properties of nanoparticles are highly dependent on the surface structure [8]. In bottom up synthesis, the nanoparticles are built from smaller entities, for example by joining atoms, molecules and smaller particles [9]. In bottom up synthesis, the nanostructured building blocks of the nanoparticles are formed first and then assembled to produce the final particle. The bottom up synthesis mostly relies on chemical and biological methods of production. [10] Synthesis is of course readily scalable, environmentally benign and compatible with the use of the product for medical applications, but production of microorganisms is often more expensive than the production of plant extracts. Plants mediated nanoparticle synthesis using whole plant extract or by living plant were also reported in literature [11]. [12]. It has been previously reported that the Graviola extracts have significant anti-cancer effects in a number of cancer cell lines both in vitro and in vivo. Studies revealed the Graviola extracts as having selective inhibition of breast cancer cells via EGFR down regulation. [13].The epidermal growth factor receptor (EGFR) is an oncogene frequently over expressed in breast cancer (BC), and its over expression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. In addition, experiments showed that Graviola fruit extract (GFE) inhibit the growth of BC cells using xenografts mouse model studies. Moreover, GFE selectively inhibited the growth of EGFR-over expressing human BC (MDA-MB-468) cells but not in non-tumorigenic human breast epithelial cells (MCF-10A). [14].These studies strengthen the evidence that Graviola has selective anti-growth effects between cancer and non-cancer cells.

 

MATERIALS AND METHODS:

SAMPLE COLLECTION:

The fruit of Annona muricata (Graviola) were collected from the market of Paris corner, Chennai.

 

PROCESSING OF Annona muricata (graviola):

The Annona muricata (pulp) were dried under shade and grinded to coarse powder and stored in well closed container.

 

EXTRACTION PROCESS:

Successive extraction was carried out with shoxlet apparatus with organic solvent ethanol.

 

SYNTHESIS OF SILVER NITRATE:

One mill molar of 95-ml silver nitrate (0.016 g) solution was prepared and kept in a 250-ml Erlenmeyer flask. Graviola fruit extract (5 ml) was added to the silver nitrate solution. Ninety-five percent of the bio reduction of AgNO3 ions occurred within 72hours. The yellow colure solution which slowly turned brown indicated the formation of silver nanoparticles.

 

UV-VISIBLE ABSORPTION SPECTROSCOPY:

The extract and the synthesized nano particles were observed to cross check the synthesis silver nano particle through absorption spectrum recorded between 400nm-500nm. The peak between this nm confirms the synthesis of silver Nano particle.

 

FOURIER TRANSFORM INFRA RED (FTIR):

Fourier Transform Infra-Red (FTIR) spectroscopy measurement, the bio-transformed product present in cell-free filtrate were freeze-dried and diluted with potassium bromide in the ratio of 1:100 FTIR spectrum of samples was recorded in Nicolet Impact 400FT-IR spectrophometer instrument with a diffuse reflectance mode (DRS 8000) attachment. All measurement were carried out in the range of 400-4000´cm at a resolution of 4cm.

 

SCANNING ELECTRON MICROSCOPE (SEM) ANALYSIS:

Scanning electron microscope (SEM) SEM technique was employed to visualize the size and shape of Ag nanoparticles. This scanning electron micrograph was taken using a Philips Scanning electron microscope. Dried powder of the silver was placed on carbon-coated copper grid.

 

ANTICANCER ACTIVITY:

IN VITRO ASSAY FOR CYTOTOXICITY ACTIVITY (MTT ASSAY)

The Cytotoxicity of samples on HT-29 was determined by the MTT assay (Mosmann et al.,1983). Cells (1 × 10⁵/well) were plated in 1ml of medium/well in 24-well plates (Costar Corning, Rochester, NY). After 48 hours incubation the cell reaches the confluence. Then, cells were incubated in the presence of various concentrations of the samples in 0.1% DMSO for 48h at 37°C. After removal of the sample solution and washing with phosphate-buffered saline (pH 7.4), 200µl/well (5mg/ml) of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl--tetrazolium bromide cells(MTT) phosphate- buffered saline solution was added. After4h incubation, 0.04M HCl/ Isopropanol was added. Viable cells were determined by the absorbance at 570nm .Measurements was performed and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The absorbance at 570 nm was measured with a UV- Spectrophotometer using wells without sample containing cells as blanks. The effect of the amples on the proliferation of HT-29 was expressed as the % cell viability, using the following

 

FORMULA:

% cell viability = A570 of treated cells / A570 of control cells.

 

RESULTS AND DISCUSSION:

Synthesis

Fig. 1: A-Sample extract without nano particle and B-Sample extract with nano particle.

 

The present study demonstrates successful green synthesis of nanoparticles by using Annona muricata pulp extract. The nanoparticles synthesised was confirmed by colour changes from pale yellow to brown (Fig1.). 

 

UV ANALYSIS:

 

Fig.2: Showing UV Absorbance (a) without nano particle at 267nm, (b) with nano particle at 448nm, (c) with diluted nano particle at 250nm.

 

Fig 2. Shows the surface plasmon resonance of the silver nanoparticles reduced by Annona muricata extract was obtained by 448nm and the observation of plant extract and diluted nanoparticle were recorded by 267nm and 250

 

FTIR- SPECTRA ANALYSIS:

 

Fig.3: IR Spectrum analysis of plant sample 

 

Fig.3: IR Spectrum analysis of Nanoparticle with plant sample

 

The IR spectral pattern also accounts for presence of alkyl groups (CH₃, CH₂) and this due to the presences of sp3 hybridised CH stretching vibration. In addition to this the presence of (C=O) carboxyl group is also observed due to the peak at 1600-1740range.

 

SEM ANALYSIS (SCANNING ELECTRON MICROSCOPE)

 

Fig.5: SEM analysis showing the size of nano particle ranged from 25.5nm-46.9nm.

Table: 1: Anticancer effect of control (without nano particle) on HT-29 cell line against Annona muricata pulp extract.

S. No	Concentration (µg/ml)	Dilutions	Absorbance (O.D)	Cell viability (%)
1	1000	Neat	0.06	13.3
2	500	1:1	0.11	24.4
3	250	1:2	0.18	40.0
4	125	1:4	0.22	48.8
5	62.5	1:8	0.25	55.5
6	31.2	1:16	0.30	66.6
7	15.6	1:32	0.34	82.9
8	7.8	1:64	0.39	86.6
9	Cell control	-	0.45	100

 

Table.2: Anticancer effect of Silver conjugate on HT-29 cell line against Annona muricata pulp extract.

S. No	Concentration (µg/ml)	Dilutions	Absorbance (O.D)	Cell viability (%)
1	1000	Neat	0.08	17.7
	500	1:1	0.13	28.8
3	250	1:2	0.17	37.7
4	125	1:4	0.21	46.6
5	62.5	1:8	0.24	53.3
6	31.2	1:16	0.28	62.2
7	15.6	1:32	0.31	68.8
8	7.8	1:64	0.35	77.7
9	Cell control	-	0.45	100

 

The in vitro anticancer activity effect of Annona muricata silver nanoparticle was tested on HT-29 cell line. The minimum inhibitory concentration was observed.

 

CONCLUSION:

In this present study silver nanoparticle was rapidly synthesized using Annona muricata pulp extract. It is simple, faster, cost effective and eco-friendly method for pharmacology research. Further the plant extract derived silver nanoparticles were recorded strong cytotoxicity effects against HT-29 cell line. However, the synthesized silver nanoparticles could have a high potential for use in the treat of anticancer. 

 

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Accepted on 20.01.2017           © RJPT All right reserved