Cytotoxicity and Antimicrobial Activities of Copper Nanoparticles Synthesised using Mimosa pudica

 

D. Malathy

Assistant Professor, School of Basic Sciences, Department of Chemistry, VISTAS, Pallavaram,

Chennai-600 117, India

 

ABSTRACT:

The present study was to evaluate the biological activity of copper nanoparticles synthesized using ethanolic extract of Mimosa pudica. The plant has been used traditionally for ages, in the treatment of urogenital disorders, piles, dysentery, sinus, and also applied on wounds. In current study, ethanolic extract of Mimosa pudica was used for the synthesis of copper nanoparticles. The formation of copper nanoparticle was confirmed by UV-Visible spectroscopy. Fourier Transform Infra red Spectroscopy (FTIR) revealed the possible involvement of photo-constituents in copper nanoparticles of crude extract.XRD is commonly used to determine the chemical composition and crystal structure of a material. Further Transmission Electron Microscopy (TEM) was used for the shape and morphology determination. The bio- synthesized copper nanoparticles were found to be effective against pathogenic bacterial and fungal strains. The in-vitro cytotoxicity activity of copper nanoparticles was performed against MCF-7cell line (breast cancer cell line) and compared against the vero cell line (normal cell line).The result showed that the copper nano particles show moderate activity against the cancer cell but seems to have less toxicity towards normal cell.

 

 

1. INTRODUCTION:

Mimosa pudica is a famous ornamental plant commonly known as sleeping grass, sensitive plant, humble plant, shy plant, touch‐me‐not, chuimui, and lajwanti among other names [1]. M. pudica is also used to avoid or cure several disorders like cancer, diabetes, hepatitis, obesity, and urinary infections [2]. M. pudica is famous for its anticancer alkaloid, mimosine, along with several valuable secondary metabolites like tannins, steroids, flavonoids, triterpenes, and glycosylflavones [3-6].

 

 

A wide array of pharmacological properties like antioxidant, antibacterial, antifungal, anti‐inflammatory, hepatoprotective, antinociceptive, anticonvulsant, antidepressant, antidiarrheal, hypolipidemic activities, diuretic, antiparasitic, antimalarial, and hypoglycemic [7-20] have been attributed to different parts of M. pudica.

 

2 MATERIALS AND METHODS:

Materials:

Mimosa Pudica, the sample for the biosynthesis of the copper nanoparticles was purchased from the local supermarket. Copper nitrate used in the present study were of high purity and analytical grade and purchased from Ranbaxy chemical company, Inc, India The solvent ethanol was re distilled before use. Double Distilled water was used for the synthesis.The bacterial strains employed in this work were procured from microbial type culture collection centre (MTCCC) located at the institute of microbial technology, Chandigarh, India. (Staphylococcusaureus, E.coil, Pseudomonas aeruginosa, Bacillus subtilis and Salmonella spp).

Preparation of sample:

Fresh leaves of mimosa pudica were thoroughly washed under running tap water followed by washing it with double distilled water to remove surface impurities. They were crushed using a blender to get powder. The dried Pulverized plant material of 5 g was extracted in 100 ml of ethanol at 60°C for 2 hours. The extracts were filtered using Whatmann No. 1 filter paper and the analysis was carried out immediately without storage for the bio synthesis of copper nanoparticles.

 

Bio synthesis of copper nanoparticles:

To 100 ml of ethanolic extract of mimosa pudica, added 90 ml of 1mm of copper nitrate solution in 250 ml beaker. The formation of copper nanoparticles is confirmed by the color change from pink to brown when it is kept on a water bath at 80°C. The formation of copper nanoparticles is inferred by visual observation followed by recording UV-Visible spectrum. The biosynthesized copper nanoparticles are characterized by FTIR, SEM and XRD studies.

 

UV-VISIBLE SPECTROSCOPY:

The reduction of copper nitrate to copper was monitored by recording UV-Visible spectrum of the reaction mixture after diluting a small aliquot of the sample with deionised water. The measurements are recorded on Shimadzu dual beam spectrometer (model uv-1650pc) operated at resolution of 1nm.

 

Fourier transform infra-red (ftir) spectroscopy:

FT-IR measurement was carried out for both the extract and copper nanoparticles to identify the possible bioactive molecules responsible for the reduction of the copper ions. FT – IR Spectra were recorded using a perkin Elmer 360 model IR double beam spectrophotometer The Spectra were collected from 4000 to 400 cm-1 with 4 cm-1 resolution over 40 scans All spectra were collected against the background spectra of KBr.

 

X-RAY Diffraction Studies:

X-ray diffraction (XRD) measurement of the copper nanoparticles was carried out using powder x-ray diffractometer instrument (SEIFERT JSO DEBYEFLEX-2002) in the angle range of 100 -700 operated at a voltage of 40Kv and a current of 30mA with CuKα radiation in a θ-2θ configuration. The crystallite domain size was calculated by using Debye- Scherrer formula.

 

Transmission electron microscopy:

TEM images were obtained with a field emission JEOL JEM-1400 120 KV instrument.

 

Anti bacterial studies:

Antibacterial of extracts was determined by disc diffusion method on Muller Hinton agar (MHA) medium. Muller Hinton Agar (MHA) medium is poured in to the petriplate. After the medium was solidified, the inoculums were spread on the solid plates with sterile swab moistened with the bacterial suspension. The disc were placed in MHA plates and add 20 µl of sample (Concentration: 1000µg, 750µg and 500 µg) were placed in the disc. The plates were incubated at 37ºC for 24 hrs. Then the antimicrobial activity was determined by measuring the diameter of zone of inhibition.

 

Antifungal studies:

Antifungal of extracts was determined by disc diffusion method on Sabouraud Dextrose agar (SDA) medium. Sabouraud Dextrose agar (SDA) medium is poured in to the petriplate. After the medium was solidified, the inoculums were spread on the solid plates with sterile swab moistened with the fungal suspension. The disc were placed in SDA plates and add 20 µl of sample (Concentration: 1000µg, 750µg and 500 µg) were placed in the disc. The plates were incubated at 37ºC for 24 hrs. Then the antimicrobial activity was determined by measuring the diameter of zone of inhibition.

 

Anticancer studies:

The anticancer activity of samples on MCF-7 was determined by the MTT assay (288). 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) solution was added. After 4h incubation, 0.04M HCl/ isopropanol were added. Viable cells were determined by the absorbance at 570nm. Measurements were 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 samples 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 × 100%

 

3 RESULTS AND DISCUSSION:

Qualitative pharmocognostic evaluation of extract:

The results of qualitative phytochemical analysis of the mimosa pudica extract are shown in table-1 which indicate the presence of carbohydrates alkaloids, proteins, amino acids, tannins, phenolic, flavonoids and steroids.

 

Table:1 Qualitative phytochemical analysis of the mimosa pudica extract

(+) Indicates the presence of chemical constituents

(-) Indicates the absence of chemical constituents

 

Visual characterization:

When the extract was mixed in the aqueous solution of copper nitrate, it started to change the color from pink to brown.The color change to brown indicates the reduction of copper nitrate and formation of copper nanoparticles. Fig 1 indicates the formation of nanoparticles.

 

Figure 1:Color change of reduced copper

 

UV-Visible studies on copper nanoparticles:

UV-Visible absorbance spectroscopy has proved to be a very useful technique for studying metal nanoparticles because the peak positions and shapes are sensitive to particle size.The surface plasmon peak of CuNp has been reported to appear at around 570nm.

 

 

 

Fourier transform infra-red spectroscopy:

FTIR spectroscopy was used to analyze the interaction between the metal and plant extract. The wavelength of light absorbed is characteristic of the chemical bond present in the chemicals by interpreting the infra-red absorption spectrum. The chemical bond in a molecule can be determined to further realize the role of plant extract in the formation of copper nano particles. The FTIR spectra for bio synthesis metals nano particles were studied. In figure 2 of copper nanoparticles, showed strong bands at 3436.5, 1652.0, 1398.0, 1066.0 and 743 cm^-1 respectively, these intense bands corresponds to the strong stretching free hydroxyl O–H of alcohols and phenols, –C=C– alkenes, C–H rock alkanes, C–O strong stretching ethers, C–H strong bending alkene.

 

XRD analysis:

A number of Bragg reflections corresponding to (111), (200) and (220) sets of lattice planes are observed, which can be indexed to face-centered cubic copper. The peaks match with the Joint Committee of powder Diffraction Standards, which further proves the formation of copper nanoparticles.

 

 

Figure 2 IR Spectra of biosynthesised copper nanoparticles

Furthermore, the average diameter of the copper nanoparticles is calculated in the range 15-30nm by Scherrer’s formula using FWHM obtained from the diffraction peaks:

 

D= 0.89λ/βcosθ

 

Where D is the mean grain size,

λ is the wavelength of copper target,

β is the FWHM of the diffraction peaks and

θ is the diffraction angle.

 

Thus XRD is commonly used to determine the chemical composition and crystal structure of a material.

 

 

Figure 3 XRD Data for copper nanoparticles

 

TRANSMISSION ELECTRON MICROSCOPY:

The size and morphology of the copper nanoparticles synthesized using mimosa pudica extract were analyzed with a transmission electron microscopy. The typical TEM microscopy and the size of distribution of copper nano particles obtained by mixing aqueous extract of plant in mimosa pudica medium were shown in the figure 4.The resultant particles essentially were very fine with the mean diameter of 20nm Further it was observed that morphology of the particles was spherical in size.

 

BIOLOGICAL ACTIVITY OF COPPER NANOPARTICLES:

ANTI BACTERIAL ACTIVITY:

Anti bacterial studies was performed on five bacterial species namely staphylococcus aureus, E.coil, pseudomonas aeruginosa, bacillus subtilis and salmonella spp. The result for the copper nano particles and commercial antibiotics used as positive control are listed in table 2 and Zone of inhibition are shown in figure 5.

 

Table:2 Anti bacterial studies of copper nanoparticles

Organisms	Zone of inhibition (mm)			Antibiotic (1mg/ml)
	Concentration(µg/ml)
	1000	750	500
Staphylococcus aureus	7	6	-	21
Escherichia coli	8	7	6	24
Pseudomonas aeruginosa	11	7	6	9
Bacillus subtilis	7	6	6	13
Salmonella spp.,	7	6	-	27

   

Figure 4: TEM image of copper particles

 

Staphylococcus aureus                       Escherichia coli

 

P.aeruginosa                          Salmonella spp

 

Bacillus subtilis

Figure 5 Antibacterial activity of biosynthesised copper nanoparticles

 

With increasing concentration of the nano particles an increase in diameter of the zone of inhibition was observed indicating that the nano particles show better antibacterial activity.The result showed the staphylococcus aureus and salmonella spp, were found to be inactive at 500ug concentration but with increase in concentration they show increased activity.

 

ANTI FUNGAL ACTIVITY:

Anti fungal studies was performed on three fungus namely Aspergillus niger, Candeda albicans and Rhizophus.The results and commercial antibiotics used as positive control are listed in table 3 and corresponding zone of inhibition are shown in figure 6.

 

Table 4 Antifungal studies of copper nanoparticles

Organisms	Zone of inhibition (mm)			Antibiotic (1mg/ml)
	Concentration(µg/ml)
	1000	750	500
Aspergillus niger	11	8	6	9
Candeda albicans	8	8	7	31
Rhizophus	10	9	6	25

 

Aspergillus niger

 

Cande da albicans

 

Rhizophus

Figure 6 Antifungal activity of biosynthesised copper nanoparticles

 

With increasing concentration of the nano particles an increase in diameter of the zone of inhibition was observation indicating that the nano particles show better antifungal activity.

 

ANTI CANCER ACTIVITY

The invitro cytotoxicity activity of copper nanoparticles was performed against MCF-7cell line (breast cancer cell line) and compared against the vero cell line (normal cell line). The result and corresponding figure are shown intable 5 and figure7.

 

Table 5 Cytotoxicity activity of copper nanoparticles

Concentration µmg/ml	Dilutions	Absorbance (O.D)		Cell Viability (%)
		Vero	MCF-7	Vero	MCF-7
1000	Neat	0.21	0.04	42.85	7.40
500	1:1	0.25	0.09	51.02	16.66
250	1:2	0.28	0.13	57.14	24.07
125	1:4	0.31	0.17	63.26	31.48
62.5	1:8	0.34	0.23	69.38	42.59
31.2	1:16	0.39	0.28	79.59	51.85
15.6	1:32	0.42	0.31	85.71	57.40
7.8	1:64	0.45	0.34	91.83	62.96
Cell Control	-	0.49	0.54	100	100

 

Figure7 Anticancer activity of biosynthesised copper nanoparticles

 

The result showed that the copper nano particles show moderate activity against the cancer cell but seems to have less toxicity towards normal cell. The selectivity index of the synthesized copper nano particles was found to be 16.This is indicative of the fact that the biosynthesized nanoparticles show considerable activity with less side effects.

 

 

 

CONCLUSION:

The present study deals with the bio extraction of copper nanoparticles from leaf extract of mimosa pudica which provides cost effective, easy and proficient way for synthesis of nanoparticles. The antimicrobial activity of cobalt nano particles were investigated. The antimicrobial and anticancer activity of cobalt nanoparticles showed that the nanoparticles have considerable activity with less side effects.

 

ACKOWLEGEMENT:

The authors are grateful to the management of VISTAS for their encouragement to carry this work. We also thank the Life tech research centre for providing biological studies.

 

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Accepted on 21.05.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4359-4364.