Biosynthesis and characterization of copper oxide nanoparticles and its anticancer activity on human colon cancer cell lines (HCT-116)

Accepted Manuscript Biosynthesis and characterization of copper oxide nanoparticles and its anticancer activity on human colon cancer cell lines (HCT-116)

V. Gnanavel, V. Palanichamy, Selvaraj Mohana Roopan PII: DOI: Reference:

S1011-1344(17)30239-7 doi: 10.1016/j.jphotobiol.2017.05.001 JPB 10817

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

21 February 2017 5 April 2017 1 May 2017

Please cite this article as: V. Gnanavel, V. Palanichamy, Selvaraj Mohana Roopan , Biosynthesis and characterization of copper oxide nanoparticles and its anticancer activity on human colon cancer cell lines (HCT-116), Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/j.jphotobiol.2017.05.001

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ACCEPTED MANUSCRIPT Biosynthesis and characterization of Copper oxide nanoparticles and its anticancer activity on human colon cancer cell lines (HCT-116) V. Gnanavela, V. Palanichamyb,* Selvaraj Mohana Roopana,* Department of Chemistry, School of Advanced Sciences, VIT University, Vellore 632014, Tamil Nadu, India

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Department of Biotechnology, School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu,

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+91

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2336;E-mail

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[email protected];

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Corresponding

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[email protected] (S. M. Roopan)

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ABSTRACT

The eco-friendly synthesis of nanoparticles through green route from plant extracts have

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renowned a wide range of application in the field of modern science, due to increased drug

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efficacy and less toxicity in the nanosized mediated drug delivery model. In the present study,

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our research groups have biosynthesized the stable and cost effective copper oxide nanoparticles (CuO NPs) from the leaves of (Ormocarpum cochinchinense) O. cochinchinense. The synthesis

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of crystalline CuO NPs from the leaf extract of O. cochinchinense were confirmed by various

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analytical techniques like UV-Visible Spectroscopy (UV-Vis), Fourier-Transform Infrared Spectroscopy (FT-IR), X-Ray Diffractometer (XRD), Scanning Electron Microscopy (SEM),

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Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction (SAED) pattern. Further the synthesized CuO NPs were screened for anticancer activity on human colon cancer cell lines (HCT-116) by MTT (3-(4,5-dimethyl-2-tiazolyl)-2,5-diphenyl-2-tetrazolium bromide) assay. The obtained result inferred that the synthesized CuO NPs demonstrated high anticancer cytotoxicity on human colon cancer cell lines (HCT-116) with IC50 value of 40 µg mL-1 were discussed briefly in this manuscript. Keywords: Ecofriendly, Ormocarpum cochinchinense, CuO NPs, Cytotoxicity 1

ACCEPTED MANUSCRIPT 1. Introduction Natural compounds obtained from the plant materials are known to be very much safer and easily metabolized drug when compared to other synthetic medicinal compounds [1]. The secondary metabolites obtained from the plant materials leads towards development of drugs

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[2].Nearly one fourth of the total medicinal compounds used by the developed countries are

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natural compounds [3].

Cancer is one of the severe causes for the increased mortality rate in many countries and also

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it sated to be dangerous health threat to our mankind worldwide, which is projected to report new cases of about 25 million per year as per World Cancer Report from World Health Organization

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[4]. The major available cancer treatments like alkylating agents, antimetabolites and other

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different cancer therapy methods have several side effects due to the incapability differentiation

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between the normal cell and cancer causing cells which leads to toxicity [5]. The application of nanomaterials in the cancer treatment has been found as an important means for discovery the

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modern cancer drugs [6]. Nanoparticles are also said to be structured modern medicines which are very much useful for the treatment of cancer disease due to its nanoscale sized property

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which provides increased drug efficacy and sustained release of drug material [7].

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The CuO NPs have unique property in the field of nanoscale technological aspect. The CuO NPs have narrow band gap of 1.7 eV which makes its application wider in the field of superconductors[8-11], solar energy transformation, synthesis of organic and inorganic nanostructure composites[12], gas sensors[13-15], magnetic resistant equipment’s[16-17], antifungal, antimicrobial, anti-biotic agents[18]. The CuO NPs are also used in pesticide research because of its biocidal characteristics [19-20] and antibacterial agent [21].

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ACCEPTED MANUSCRIPT CuO NPs can be achieved by microwave irradiations [22], sol-gel method [23], electrochemical technique[24], thermal decomposition [25]and alkoxide supported method [26].These techniques have many drawbacks mainly because of employing harmful chemicals, high energy utilization and difficulties in the purification of nanoparticles[27]. The process of toxic free green materials

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like plant extract and microorganisms [28-38] for the preparation of nanoparticles provides a

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choice of advantages for the application in pharmaceutical and drug discovery safety because of

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toxic free chemicals procedure in the nanoparticles preparations. O. cochinchinense (L.) belongs to the family Fabaceae has been widely used by the traditional healers for the management of

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bone fracture disorders and its associated diseases in the region around Vellore District of Tamil

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Nadu, India [39].The present work was intended to prepare the novel, toxic free, rapid and cost management effective method for the synthesis of CuO NPs with

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2.1. Materials

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2. Materials and Methods

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cochinchinense leaf extract.

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The fresh leaves of O. cochinchinense were collected from the Vellore District of Tamil Nadu, India (12°56’0” N, 79°8’0” E) and the collected plant materials were authenticated by

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Prof. Jayaraman, PARC, Chennai, Tamil Nadu, India (No. PARC-2012-1391).Copper chloride (CuCl2) and ethanol were obtained from Sigma Aldrich Chemicals Ltd., Mumbai, India. Double distilled water was used right through the experiment. 2.2. Preparation of O. cochinchinense leaf extract The fresh leaves of O. cochinchinense were washed in running tap water to remove the filth and dust. The hygienic leaves materials were dried over the shadow in a room temperature for

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ACCEPTED MANUSCRIPT about 72 hours. The dried leaves materials were made into fine particles by using the mechanical grinder. The plant materials were extracted with ethanol by Soxhlet apparatus for 4 hours and subjected to rotary evaporator to remove the excess solvent. The concentrated ethanolic leaves extract of O. cochinchinense were filtered and collected for further process.

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2.3. Preparation of CuO nanoparticles

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About 50 mL of freshly prepared 0.003M hydrated CuCl2 solution was mixed with 50 mL of ethanolic leaves extract of O. cochinchinense and boiled for three hours at 60°C with continuous

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stirring. The formations of CuO NPs were monitored by UV- Visible spectroscopy. Due to the surface plasmon resonance excitation, the color change from yellow to brown is observed which

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indicates the formation of CuO NPs. After the color change indication observed, the prepared

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content was centrifuged at 3000 rpm for 20 mins. The pellet formed was washed with distilled

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water and kept in furnace using crucible at 400° C for 2 hr. 2.4. Characterization of CuO nanoparticles

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The formation of O. cochinchinense mediated CuO NPs was confirmed by processing

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various analytical techniques like UV-Visible Spectroscopy (Shimadzu UV-1800 PC, Japan) between the wavelength of 200-800 nm [40]. The X-Ray Diffractometer (XRD) analysis for the

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synthesized CuO NPs were done by using Advance Power XRD, model D8 (Bruker, Germany). The Scherrer formula was used to calculate the particle size determination using the formula, D = Kλ/βCosθ where D is the particle size, λ is the wavelength, K is a Scherrer constant having value of 0.94, β is a half width maximum and θ is the diffraction angle. The functional group present in the synthesized CuO NPs was identified by using FT-IR Spectroscopy (Bruker, Germany) between the wave number of 400 cm-1 to 4000 cm-1. The shape of the synthesized CuO NPs was determined by using Scanning Electron Microscope (JSM – 6390 LV Model, JEOL). The size of 4

ACCEPTED MANUSCRIPT the synthesized O. cochinchinense mediated CuO NPs was determined by Transmission Electron Microscope (JEOL, TEM-1230, USA). 2.5. Cytotoxicity study of CuO nanoparticles on human colon cancer cell line (HCT-116) The cytotoxic effect of the O. cochinchinense mediated synthesized CuO NPs against

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human colon cancer cell line (HCT-116) was done by MTT assay. Approximately thousands and

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thousands of human colon cancer cells were allowed to fix in 96 well plates at 37°C. The different concentrations of CuO NPs synthesized from leaves of O. cochinchinense (10, 20, 40,

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60, 80 and 100 µg mL-1) were treated with cancer cells after 24 hours. The drug free medium was used to wash the cells after the drug treatment. After the completion of drug treatment

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incubation period, 10 µL of 5-diphenyltetrazolium bromide (5mg/mL in PBS, MTT) and 10 µL

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of 3-(4,5-dimethylthiazol-2-yl)-2 were added to each well and incubated for four hours at 37°C

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followed by the addition of 100 µL of 0.04 mol/L hydrochloric acid in isopropanol. Absorbances were recorded at the wave length of 570 nm and 630 nm for both the test and reference was

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measured using ELISA plate reader. The plot was made for the percentage of survival cells at

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every different concentration to the group and control used [41, 42]. 3. Results and Discussion

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3.1. UV-Visible Spectral analysis of CuO nanoparticles from O. cochinchinense The UV spectral analyses for the synthesized CuO NPs were done at regular interval of time. The color change from yellow to brown color primarily indicates the conversion of Cu into CuO NPs. The UV spectrum of the synthesized CuO NPs evidently indicates the progression and stability as shown in the Fig. 1. The surface plasmon resonances of the nanosized copper oxide particles were confirmed by the appearance of maximum absorbance at the 200 nm.

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ACCEPTED MANUSCRIPT 3.2. FT-IR analysis of CuO nanoparticles from O. cochinchinense The FT-IR transmittance analysis was carried out to determine the functional groups present in the synthesized copper oxide nanoparticles from O. cochinchinense. The FT-IR analysis shows different characteristics peaks at 476.42cm-1, 603.72cm-1, 848.62cm-1, 1338.60cm-1,

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1550.77cm-1, 3323.35cm-1 between the range 400 – 4000cm-1 (Fig. 2). The slight broad band at

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3323.35cm-1 corresponds to the N-H stretch due to amine group and the peak at 1550.77cm-1

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shows the presence C=C bending due to the presence of aromatic secondary metabolites. The

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band at 1338.60cm-1corresponds to the presence of C-N stretch due to amine group and the peak at 848.62cm-1shows the presence of =C-H bending due to the alkene group. The prominent peak

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at 476.42cm-1 confirms the presence the Cu-O vibration in the synthesized CuO NPs [43].

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3.3. XRD, SEM and TEM analysis of CuO nanoparticles from O. cochinchinense

O. cochinchinense CuO NPs to confirm the crystalline nature of synthesized nanoparticles (Fig.

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3). The XRD analysis of the CuO NPs was interrelated with the Joint Committee on Powder

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Diffraction Standards (JCPDS), which confirmed the crystalline nature of CuO NPs (JCPDS 96901-5925).The morphological studies for the synthesized O. cochinchinense mediated CuO NPs

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were ascertained by SEM and TEM analysis. The Scanning Electron Microscope (SEM) analysis for the synthesized nanoparticles indicated the formation of CuO NPs around 2 µm and 1µmin an agglomerated cluster forms as shown in the (Fig.4 A-B). The Transmission Electron Microscope (TEM) analysis for the synthesized nanoparticles reported the crystalline nature of CuO NPs in agglomerated cluster structure as depicted in the (Fig. 5 A-D). The crystalline nature of the synthesized CuO NPs were confirmed by the Selected Area Electron Diffraction (SAED)

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ACCEPTED MANUSCRIPT indicated the formation of intermittent dots on the concentric circles in the SAED pattern as shown in the Fig. 6. 3.4. Cytotoxicity of CuO nanoparticles from O. cochinchinense The cytotoxicity effect of O. cochinchinense mediated CuO NPs on human colon cancer cell

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line (HCT-116) was studied by MTT assay. The varied concentrations from 10, 20, 40, 60, 80

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and 100 µg mL-1 were used for the cytotoxicity effect of O. cochinchinense mediated CuO NPs on human colon cancer cell line as shown in the Fig. 7. The synthesized copper oxide

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nanoparticles have proved significant cytotoxicity effect on human colon cancer cell line with IC50 value of 40 µg mL-1 (Fig. 8 A-B). The percentage of cell viability was declined to 22%

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when the concentration of the CuO NPs was gradually increased to 100 µg mL-1 on human colon

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cancer cell line.

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4. Conclusion

The present study has evidently demonstrated that the leaves of O. cochinchinense can be an

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unconventional resource for the green synthesis of CuO NPs. The nature of the synthesized CuO

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NPs was characterized by the analytical techniques like UV-Vis, XRD, SEM, TEM and SAED. The CuO NPs synthesized from the leaves of O. cochinchinense have proved the substantial

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anticancer activity on human colon cancer cell line with IC50 value of 40 µg mL-1. Further, identification and isolation of the biologically active compound from the leaves extract of O. cochinchinense would pave the way for discovery of novel anticancer drug. The superior efficacy and lesser toxicity of novel anticancer drug are mainly due to the application of nanosized CuO based drug designing molecules.

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ACCEPTED MANUSCRIPT Conflicts of Interest The authors confirm that there are no known conflicts of interest associated with this publication.

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Acknowledgements

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The authors express sincere thankfulness to VIT University for providing the excellent

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lab facility to carry out the experiment. We also express thanks to Prof. Jayaraman, PARC,

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Figure captions

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Fig. 1. UV-Visible spectrum of the synthesized CuO NPs

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Fig. 2. FT-IR spectrum of the synthesized CuO NPs Fig. 3. XRD pattern of synthesized CuO NPs

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Fig. 4. (A-B) SEM images of synthesized CuO NPs

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Fig. 5. (A-D)TEM images of synthesized CuO NPs

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Fig. 6. SAED image of synthesized CuO NPs Fig. 7. Cytotoxicity study of CuO NPs on human colon cancer line (HCT-116) Fig. 8. Morphological study of cancer cell lines (A - HCT 116 Control, B - HCT 116 Treated)

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ACCEPTED MANUSCRIPT Highlights Eco-friendly approach for the synthesis of CuO nanoparticles.

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Leaves extract of O. cochinchinense was used as green source.

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Green synthesized CuO nanoparticles showed significant anticancer cytotoxicity activity.

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