int j pharm sci nanotech vol 8; issue 4 international ... · and zeta potential, sem and the...

3018 Int J Pharm Sci Nanotech Vol 8; Issue 4 October December 2015

Research Paper

Anticancer Activity of Zinc Nanoparticles Made using Terpenoids from Aqueous Leaf Extract of Andrographis Paniculata M. Dhamodarana* and S. Kavithab aDepartment of Chemistry, Peruntalaivar Kamarajar Institute of Engineering and Technology (PKIET), (Government of Puducherry Institution), Karaikal, Puducherry U.T, India; and bResearch and Development Centre, Bharathiar University, Coimbatore - 641 046, India.

Received May 28, 2015; accepted July 5, 2015

ABSTRACT

In recent years, the evolution of green chemistry in the production of nanoparticles has wrapped up an immense consideration because traces of chemicals left unreacted in the chemical synthesis process can be precarious. Green synthesis of metal nanoparticles is an interesting issue of the nanoscience and nanobiotechnology. There is a growing attention to biosynthesis the metal nanoparticles using organisms. Among these organisms, plants seem to be the best candidate and they are suitable for large scale biosynthesis of nanoparticles. Nanoparticles produced by plants are more stable, and the rate of synthesis is faster than that in the case of other organisms. Natural products, especially of plant origin, represent an excellent starting point for research. In traditional medicine there are also several plants that are used to treat many diseases. Therefore, a competent protocol for the production of Zn- NPs without calcinations was developed by green

synthesis method using one of the major constituents, terpenoids from aqueous leaf extracts of Andrographis paniculata. Among the single compounds extracted from Andrographis paniculata, andrographolide is the major one in terms of bioactive properties and abundance. The anticancer activities of Zn-TAP NPs have been evaluated in cancer models such as HeLa, Hep-2 cells and were examined in different concentrations by MTT assay method. The Zn-TAP NPs showed a maximum activity against HeLa (human cervical cancer cells) and Hep-2 (human liver cancer cells) with maximal inhibition of 59% and 63% at 250 μg/ml, respectively. This approach offers environmentally beneficial alternatives to more hazardous chemicals and processes and promotes pollution prevention by the production of nanoparticle in their natural environs.

KEYWORDS: Nanoparticle; nanoscience; nanobiotechnology; A.paniculata; andrographolide; Zinc particles; HeLa cells.

Introduction

Globally, cancer represents a substantial burden of disease in the community. Every year over 200,000 people are diagnosed with cancer in the United Kingdom only, and approximately 120,000 die as an aftermath of the disease (Department of Health 2000). According to the international Agency for Research on cancer 2002, Cancer killed > 6.7 million people around the world and another 10.9 million new cases were diagnosed (Newman et al., 2003; Regina et al., 2014). It’s time people stopped living in the illusion that they can’t be afflicted with cancer. Cancer is in the climbing range in the Indian graph due to rapid lifestyle changes, adding nearly a million new cases every year.

We have one million new cancer cases coming up every year in India. In the last decade, lifestyle related causes have increased our susceptibility to the disease (AIIMS, 2013). Over the past decades, herbal drugs have been accepted universally for the treatment of a range of human diseases. Conventional medicine is widely used in

many developing countries like India. Use of herbal products has increased dramatically in the last two decades (Akoachere et al., 2002; Alarmal Mangai et al., 2014). According to WHO, 80% of world’s health problems are treated by herbal drugs only (Etkin, 1981; WHO, 2003). Medicinal plants can be promising source of noval chemotherapeutic agents including cancer. Out of 25,000 plant species existing on the earth, approximately one thousand species have anticancer potential (Yamin et al., 2002). A large number of plant species have been screened through bioassays for the search of noval herbal anticancer drugs (AbuDahab and Afifi, 2007). Several medicinal plants all over the world are being used traditionally for the prevention and treatment of cancer. However, only few medicinal plants have attracted the interest of scientist in investigating the remedy for neoplasm (tumour or cancer) (Adhuri and Pandey, 2009).

Nanotechnology is an upcoming and hasty mounting field of science which is being exploited in an extensive spectrum of disciplines such as electronics, energy,

International Journal of Pharmaceutical Sciences and NanotechnologyVolume 8Issue 4 October – December 2015

MS ID: IJPSN-5-28-15-DAMODHARAN

3018

Dhamodaran and Kavitha: Anticancer Activity of Zinc Nanoparticles made using Terpenoids 3019

environment and health sectors. Nanoscience has revolutionized these fields in achieving the processes and products that are hardly possible to evolve through conservative systems. Zinc oxide nanoparticles are presently under intensive study for applications in the field of optical devices, sensors, catalysis, biotechnology, DNA labeling, drug delivery, medical, chemical and biological sensors and as catalyst. Nanosized ZnO has been used in sun screen coatings and paints because of its high UV absorption efficiency and transparency to visible light (Fan and Lu, 2005; Gnanasangeetha et al., 2014).

Increasing awareness towards green chemistry and biological processes has led to the development of an eco-friendly approach for the synthesis of nanoparticles. The plant phytochemical with antioxidant property is accountable for the preparation of Zinc oxide nanoparticle. To pursue a healthy life and space it is imperative to develop a green synthetic approach to obtain nanomaterials targeted on different applications. It is dazzling to profuse interest this research opens with a short course on how to synthesize Zinc oxide nanoparticle in a natural scale (Gnanasangeetha et al., 2013).

The plant species Andrographis paniculata popularly known as Nilavembu, belongs to the family Acanthaceae, is distributed throughout the South India, Asian countries and Sri Lanka. The important preparations using the drug are Tiktakagheta, Gorocandi gulika, Candanasava, and Panchatiktam kasaya (Hosamani et al., 2011; Sivarajan, 1994). A preparation called “Alui” is prepared by mixing powdered cumin (Cuminium cyminum) and large cardamom (Amomum subulatum) in the juice of this plant and administered for the treatment of malaria (Thakur et al., 1989). It is known as “King of bitters”. It is the source of several diterpenoids of which the bitter water soluble lactone andrographolidic properties. As medicinal plants are gaining more importance in Pharmaceutical industries for the preparation of new phytomedicines, this study was undertaken to check its properties as a drug (Sule et al., 2010). A. paniculata species have been shown to contain compounds with significant activity and its extract has antimicrobial, antioxidant and anti inflammatory, anti parasitic, antihyperglycemic, hypoglycemic and antiallergic properties. Ethanolic extract of Andrographis paniculata possessed significant anticancer activity and also reduce peroxidation levels due to higher content of terpenoids and flavonoids. It also stimulates the central nervous system and is cytotoxic for tumor cell activities.

Therefore, this study was undertaken to prepare Zn- TAP NPs using A. paniculata with a goal of aiding the developing of new anticancer drugs with increased efficiency. We have synthesized Zn- TAP NPs using aqueous extract of A.paniculata and it was confirmed by colour transformation and UV-visible spectrophotometry. The size of nanoparticles were observed by particle size, and Zeta potential, SEM and the stability of nanoparticles was studued by FTIR. The synthesised Zn- TAP NPs over human pathogens such as HeLa, Hep-2

were studied in the anticancer activity by in-vitro MTT assay method.

Materials and Methods

Collection of plant, chemical materials & cancer cell lines

The plant Andrographis paniculata was collected from the campus of A.V.C. Arts & Science College, Mayiladuthurai. Zinc nitrate, Sodium hydroxide, alcohol, MTT and DMSO (all are of A.R grade in Merck chemicals) were purchased from Ponmani & Co, Trichy. The human cervical cancer cell line (HeLa) and laryngeal epithelial human cell line (HEP-2) was obtained from National Centre for Cell Science (NCCS), Pune.

Preparation of the extract

Andrographis paniculata leaves were collected and washed with tap water then rinsed with distilled water, dried, cut into fine pieces and were crushed into fine powder and stored at 37o C.

Preparation of Solvent

10 grams of sample powder was placed in 100 ml of ethanol and kept at room temperature for 7 days. The extract was filtered through a sterile funnel containing sterile Whatmann filter paper No.1 and filtered. It contains preservatives and stored in a brown bottle at 4°C.

Separation of TAP

The TAP was separated by column chromatography method. In this method, 25 gm silica gel powder was filled in column apparatus and poured ethanol up to slurry form then the solvent was completely eluted, and added 50% mixture (65 ml CHCl3 and 5 ml MeOH ) was added followed by 10 ml ethanolic sample. Finally remaining mixture was added. The solvent is eluted. After 12 hrs TAP was collected in other test tube.

Synthesis of Zinc Nanoparticles (Zn-NPS)

ZnO nanostructures were prepared by co-precipitation method. 0.1N aqueous solution of zinc nitrate (Zn (NO3)2·4H2O) was put into 50 ml of distilled water under vigorous stirring. After 10 min stirring, extract was taken in the ratio of 1.0 ml was added into the above solution. After addition of extract, 0.1N NaOH aqueous solution was introduced into the above aqueous solution, resulting in a white aqueous solution at pH 12, which were then placed on magnetic stirrer for stirring at 2 hr. The precipitate was then taken out and washed repeatedly with distilled water followed by ethanol to remove the impurities for the final products. Then a white powder was obtained after drying at 60 oC in vacuum at oven overnight. The whole mode of proposed method for the synthesis of Zn-NPs mediated by aqueous extract was illustrated.

Cancer cell line

The HeLa and HEP-2 cell lines are grown in Eagles Minimum Essential Medium (EMEM) containing 10%


fetal bovine serum (FBS) (Mosmann et al., 1983; Monks et al., 1991; Wilson, 2000). All cells were maintained at 37 oC, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

Cell treatment procedure

The HeLa and Hep2 monolayer cells were detached with trypsin-ethylene diamine tetra acetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 4 × 104 cells/ml in a volume of 0.1 ml. One hundred microlitres per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 370C, 5% CO2, 95% air and 100% relative humidity. After 24 hr the cells were treated with serial concentrations (50–250 mg/ml) of the test samples. They were initially dissolved in dimethylsulfoxide (DMSO) and diluted to twice the desired final maximum test concentration with serum free medium. Additional four, 2 fold serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 μl of these different sample dilutions were added to the appropriate wells already containing 100 μl of medium, resulted the required final sample concentrations. Following drug addition, the plates were incubated for an additional 48 hr at 37 oC, 5% CO2, 95% air and 100% relative humidity. The medium containing without samples were served as control and triplicate was maintained for all concentrations.

MTT Assay

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is a yellow coloured water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore, the amount of formazan produced was directly proportional to the number of viable cells which was measured spectro photometrically (Chavhan et al., 2010; Freshney, 2000). Since the reduction in MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. 5-Fluorouracil was used as a positive control. Control cells were treated with the highest concentration of DMSO (0.1%) as vehicle control. After 48 hr of incubation, 15 μl of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4 hr. The medium with MTT was then flicked off and the formed formazan crystals are solubilized in 100 μl of DMSO and then measured the absorbance at 570 nm using micro plate ELISA reader (Spectra MAX 190, Molecular Devices Corporation, USA).

The anticancer activities of the Zn-NPs against HeLa, Hep2, cell lines by using MTT assay was carried out (Freshney, 2000). MTT assay method is widely used method for the detection of cell survival and growth.

After 24 hr, the silver nanoparticle (50–250 mg/ml) which was dissolved in a medium was added to each well and incubated for 48 hr at 37 °C in a CO2 incubator. After the incubation, MTT solution (100 µl/well, 1 mg/ml) was added to each well and incubated again for 4 hr. The culture media were then removed and 100 µl of DMSO was added to each well for 1 hr. Absorbance at 570 nm was detected by microplate ELISA reader (Spectra MAX 190, Molecular Devices Corporation, USA). The % cell inhibition was determined using the following formula (Kohler, 1975; Maniatis et al., 1978; Mosmann et al., 1983).

% cell Inhibition = 100 – Abs (sample)/Abs (control) ×100

Statistical analysis

The results of the study were based on experimental values that were performed in triplicate. The statistical analysis was performed using the analysis of variance (ANOVA) to determine the effect of extracts concentration on treated cells cytoviability. Non linear regression graph was plotted between % Cell inhibition and Log10 concentration. The IC50 was determined using Graph Pad Prism software (version 3.00).

Cytotoxicity of Zn-Np’s

The Zinc nanoparticle from TAP was initially evaluated for their effects on cell viability through cytotoxic test. Cytotoxic effect of Zinc nanoparticles was observed the IC50 value was calculated as 300µg/ml.

Results and Discussion

Green Synthesis of Zinc Oxide Nanoparticles

ZnO nanostructures were prepared by co-precipitation method. 0.1N aqueous solution of zinc nitrate (Zn (NO3)2·4H2O) was put into 50 ml of distilled water under vigorous stirring. After 10 min stirring, sample (TAP) was taken in 1.0 ml added into the above solution. After addition of extract, 0.1N NaOH aqueous solution was introduced into the above aqueous solution, resulting in a white aqueous solution at pH 12, which were then placed on magnetic stirrer for stirring at 2 hrs. The precipitate was then taken out and washed repeatedly with distilled water followed by ethanol to remove the impurities for the final products. Then a white powder was obtained after drying at 60 oC in vacuum at oven overnight. The whole mode of proposed method for the synthesis of ZnO NPs mediated by aqueous extract was illustrated. The fig- 1 shows the schematic diagram of Zn-NPs synthesis and fig-2 are active compound (TAP) in Zn-NPs synthesis.

Fig. 1. Schematic Diagram of Zn-NPs synthesis.


Fig. 2. Active compound (TAP) in Zn-NPs synthesis.

Figure 3 shows the colour intensity of aqueous extract of TAP incubated with Zinc nitrate solution in the beginning (a) and after 2 hr (b) of reaction. Figure (3b) revealed the bio reduction of Zn+ ions to Zn nano particles by ingredients of TAP. The synthesised Zinc nano particles maximum absorption range was measured using FTIR. The TAP Zinc nanoparticles were found to exhibit very strong absorption peaks at 466.77 nm. It clearly indicated the presence of Zinc nanopartilces.

Fig. 3a. Zinc Nitrate of TAP solution in the beginning.

Fig. 3b. Bio-reduction of Zn+ ions to Zn. Anti Cancer activity

The viability of HeLa and Hep-2 cancer cells with TAP Zn-NPs for 72 hr was determined using the colorimetric MTT-based assay. The TAP Zn -NPs exhibited a dose-dependent activity within the concentration range of 25-250 µg/ml (“Table1”and “Fig. 4a & 4b”). The TAP Zn-NPs showed a maximum activity against HeLa and Hep-2 and it was recorded as 59.10 and 62.40% at 250 µg/ml respectively.

TABLE 1

Anticancer activity of zinc oxide nonoparticles (zn-TAP).

Cell lines

Name of the

sample (%) Percentage of inhibition/Concentration µg/ml

Control 50 100 150 200 250

HeLa

Zinc Oxide Nano-particles

99.90 13.49 19.30 31.80 43.01 59.10

Hep2 99.90 18.54 28.59 35.32 46.92 62.40

The following “5-a, 5-b & 5-c figure (photos)” clearly indicate the anticancer activity and its cytotoxicity in 50 to 250 µg/ml of TAP Zn-NPs from andrographic paniculata against HeLa and Hep2 cell line.

Control Zinc nanoparticles treated cells (zn-TAP)

(a) (b)

Fig. 4. Cytotoxicity effect of TAP Zinc nano particles from andrographic paniculata against Vero (African green monkey kidney) cell line.


Control 50 µg/ml 100 µg/ml

150µg/ml 200 µg/ml 250 µg/ml

Fig. 5(a). Anticancer activity of TAP Zn-NPs against HeLa cell line.

Control 50 µg/ml 100 µg/ml

Fig. 5(b). Anticancer activity of TAP Zn-NPs against Hep-2 cell line.

150 µg/ml 200 µg/ml 250 µg/ml

Fig. 5(c). Anticancer activity of TAP Zn-NPs from andrographis paniculata against HeLa and Hep-2 cell line.

Conclusions

The anticancer activity of TAP of andrographic paniculata in the synthesis of Zinc oxide nanoparticles was evaluated. The biosynthesised TAP Zn-NPs displayed good anticancer activity over tested human pathogens HeLa and Hep-2 and it was recorded as 59.10 and 62.40% at 250 µg/ml respectively. From the datas good activity in Hep-2 cell line than HeLa. The obtained TAP Zn-NPs have potential applications in the medical field and this simple product has several advantages such as low quantity, low cost, high effectiveness, compatibility for medical and pharmaceutical applications as well as large scale production.

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Address correspondence to: M. Dhamodaran, Department of Chemistry, Peruntalaivar Kamarajar Institute of Engineering and Technology (PKIET), (Government of Puducherry Institution), Karaikal, Puducherry, India. E-mail: [email protected]

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