ISSN   0974-3618  (Print)                    www.rjptonline.org

            0974-360X (Online)

 

 

RESEARCH ARTICLE

 

Synthesis, Characterization and Antimicrobial Activities of Turmeric Curcumin and Curcumin Stabilized Zinc Nanoparticles - A Green Approach

 

Jayandran. M1, Muhamed Haneefa. M2*, Balasubramanian.V2

1Faculty of Chemistry, Mahendra Engineering College, Namakkal, India

2Faculty of Chemistry, AMET University, Chennai, India

*Corresponding Author E-mail: honey79101@gmail.com

 

ABSTRACT:

Metal nanoparticles are versatile platforms for biomedical applications and therapeutic intervention. But there is a need to develop new method in the preparation of nanoparticles which should not be harmful to environment. Recent studies demonstrated that several metal nanoparticles synthesized by green methodologies have shown potential antibacterial, antifungal activities. This paper investigates the synthesis of turmeric curcumin as well as zinc nanoparticles in the greener route by using natural lemon extract as a reducing agent and curcumin as a stabilizing agent. The obtained curcumin was confirmed by UV-Vis, IR analysis and Zn nanoparticles were characterized by UV-Vis, IR, XRD, SEM and TEM techniques. The experimental results displayed that the Zn nanoparticles with an average diameter of about 23-46 nm. The antimicrobial activity of curcumin stabilized Zn nanoparticles exhibited significant effects against all the bacterial and fungal species. But sensitivity to nanoparticles was found to vary depending on the microbial species.

 

KEYWORDS: Metal nanoparticles, Green method, Curcumin, Soxhlet extract, Antimicrobial, Therapeutic agent.

 

 


INTRODUCTION:

New technologies often create new challenges to science in addition to their benefits, raise concerns about health and various environmental problems. Recently, nanotechnology holds a promise and a broad aspect towards wide applications of nanoparticles in a multiple way of emerging fields of science and technology which referring at the nanoscale, i.e. 1 to 100 nm. Metal nanoparticles are being given considerable attention in nanoscale science and engineering technology over the last decades due to their interesting properties and potential applications in many areas of industry. Nanoparticles possess high surface to volume ratio due to its small size, which gives very distinctive features to nanoparticles1-3.

 

 

 

Received on 03.03.2015       Modified on 19.03.2015

Accepted on 25.03.2015      © RJPT All right reserved

Research J. Pharm. and Tech. 8(4): April, 2015; Page 445-451

DOI: 10.5958/0974-360X.2015.00075.X

 

Metal nanoparticles have been widely used for various applications like catalysis, optoelectronics, magnetic, thermal, sensors, fine chemical synthesis, solar energy conversion and medicine etc. The vast applications of nanoparticles in medical sciences are drug delivery, imaging, tissue repair, immune assay and diagnosis4-5. Mostly nanoparticles have been synthesized using a variety of techniques typically characterized as either a physical or a chemical method. Physical methods are capable of producing a wide range of metallic nanoparticles; however the quality of the material is not as high as chemically synthesized materials. In chemical synthesis techniques, the growth and assembly of metallic nanoparticles is controlled by optimizing reaction parameters6-9. Although chemical and physical methods may successfully produce pure, well-defined nanoparticles, these are quite expensive and possibly dangerous to the environment. At present, there is an emergent need to develop environmentally benevolent nanoparticles synthesis routes, which can be proceeded by biological method instead of using toxic    chemicals10-11. Nanomaterials are the leading in the field of nanomedicine and in that respect nanotoxicology research is gaining great importance. Accordingly, the researchers in the field of nanoparticles synthesis and assembly have turned to biological systems for inspiration. The green synthesis method plays a significant role on the effective consumptions of nanoparticles12-13. Proper utilization of environmentally benevolent solvents and nontoxic chemicals are some of the key issues in green in synthesis approach deliberations14.In the green-nanotechnology, various metal nanoparticle synthesis have been reported using microorganisms, plant extracts and other biological natural materials. Use of natural plant extracts in the preparation of nanoparticles by greener route provides advancement over chemical and physical method as it is cost effective, environment friendly15-16.

 

Curcumin is an active component of turmeric plant; it is responsible for its characteristic yellow color and therapeutic potential17. Curcumin is of considerable interest because of its antioxidant18-19, anti-inflammatory20, antimicrobial21 and anticancer activities22 etc. But its poor bioavailability remains a major challenge due to the presence of olefinic groups in its structure this β-diketone of poor aqueous solubility rendering it of relatively low bioavailability23. In order to improve the bioavailability of curcumin, various approaches have been used. One of the possible approaches to increase the bioavailability of curcumin is its conjugation on the surface of metal nanoparticles24-25.

 

Zinc nanoparticles (ZNPs) have wide applications in optoelectronics, field emission, vacuum fluorescent, luminescent light, catalysis and also photo degradation etc.26-27.Various researches have shown that antimicrobial formulations in the form of nanoparticles could be used as potential bactericidal materials. The emergence of nanoscience and nanotechnology in the last decade presents opportunities for exploring the antimicrobial effect of Zn nanoparticles. The antimicrobial effect of Zn nanoparticles has been attributed to their small size and high surface to volume ratio, which allows them to interact closely with microbial membranes and is not merely due to the release of metal ions in solution. Furthermore, Zn nanoparticle appears to effectively resist microorganisms and much more stable and has a longer life than organic based disinfectants and antimicrobial agents. Zinc is an essential constituent for cell growth and in inhibiting bacterial enzymes like dehydrogenase and certain protective enzymes such as thiolperoxidase and glutathione reductase28-30. But the surface functionalization of ZNPs with curcumin may give a new way of using the curcuminoids with Zn nanoparticles towards highly improved antimicrobial activity.

Hence, in this work we report the synthesis of curcumin from turmeric (Scheme 1) as well as the synthesis of the zinc nanoparticles by using lemon extract as reducer and curcumin as stabilizing agent (Scheme 2) to enhance the solubility of curcumin in water and its biological activities. The Zn nanoparticles functionalized with curcumin showed excellent inhibition activity against the microbial strains tested over all.

 

MATERIAL AND METHODS:

All the chemicals and solvents used were of analytical reagent grade and obtained from Merck (Indi) Ltd and all samples were prepared by using fresh double-distilled water throughout the experiment. Curcumin was isolated from turmeric (BSR-01) which was purchased from Agricultural College and Research Institute, Madurai.

 

Collection of extracts:

Lemon fruits were collected from the local markets. They were washed in double distilled water, cut into pieces and squeezed well to make 5 to 10 ml pure extract. The extract was then filtered using Whatman’s No. 1 filter paper. The filtrate was collected in a clean and dried container and it was stored for further uses. 

 

Isolation of curcumin (CR) from turmeric:

Curcumin was quantitatively extracted from turmeric in soxhlet apparatus by using 95% ethanol as per our previous work31 and the curcumin content was estimated by Manjunath et al., 199132. The high curcumin yield turmeric variety, BSR-01 was incorporated in this method for better result. The brief process is described as follows. Turmeric dried powder weighed (5 g) and taken in soxhlet apparatus and 250 ml of ethanol was poured into the apparatus. The extraction process was carried out for 2-3 hour and the final curcumin extract absorbance was measured at 425 nm against alcohol blank and the curcumin percentage was calculated. The above ethanol residual extract was evaporated and dried then recrystallized by 95% ethanol for further uses.

 

CHARACTERIZATION:

The UV-Visible absorption spectra of the samples were measured on a Shimadzu UV-Vis V-530A spectrophotometer in the range of 425nm. The nanoparticles were examined for FT-IR spectra analysis and recorded on a Jasco FT-IR/4100 spectrophotometer with 4 cm-1 resolution in the range of 4000 to 400 cm-1. X-ray measurement of the prepared solids was carried out using a Panalytical X’Pert Powder X’Celerator Diffractometer (Netherland) in the range of 10ο to 80ο 2θ of 2ο min-1. Scanning electron microscopy (SEM) images were recorded by using a JEOL Model JSM - 6390LV scanning electron microscope. High resolution transmission electron microscopy (HRTEM) was carried out using a 300 kV JEOL-3011 instrument to determine the morphological changes.

Synthesis of Zinc nanoparticles (ZNPs):

1mM aqueous solution of zinc acetate was prepared and used for the synthesis of ZNPs. Double distilled water has been used throughout the synthesis process. The filtered stored pure lemon extract (10 ml) was taken in a beaker and freshly prepared zinc solution (10 ml) was mixed with the extract with constant stirring for the reduction of zinc ions. The reaction mixture was kept in the magnetic hot stirrer at 50-600C for a particular time to color change from pale green to colorless which indicated the metal ion reduction. Then freshly prepared curcumin extract (1mM) was added with above solution mixture for stabilizing the nanoparticle and the solution color was changed to yellowish. The stirring was continued for about an hour and there was observed the color change from yellowish to yellowish brown slowly and finally a permanent dark brown color which showed the complete stabilized ZNPs. The pH was maintained between 3- 4 throughout the experiment for better result by using weak HCl and NaOH. The solution was centrifuged with washing several times with water and ethanol to remove the unreacted materials. Finally the pure ZNPs was obtained by decantingthe supernatant and kept in oven to dryness (Scheme 2).

 

Biological assay:

The antibacterial activity of the synthesized ZNPs were tested by disc diffusion method against two gram positive bacteria (Staphylococcus aureus and Bacillus subtilis), two gram negative bacteria (Escherichia coli and Staphylococcus bacillus) and antifungal activity was carried out by agar well diffusion method againstfour funguses (Candida albicans, Curvularia lunata, Aspergillus niger and Trichophyton simii). For disc diffusion method, stock cultures incubated in nutrient agar were transferred to test tube of Muller-Hinton broth (MHB) for bacteria that were incubated for 24 hour at 37oC. The cultures were diluted with fresh Muller-Hinton broth to get 2.0Χ106 CFU/ml for bacteria. The Muller Hinton Agar (MHA) plates were prepared by pouring 15 ml of molten media into sterile petri plates. The sample was loaded placed on the surface of the cultured agar plates and incubated at 37oC for 24 hours then inhibition zones formed around the disc were measured and the results were compared with standard antibiotic, Chloramphenicol. For agar well diffusion method, the fungal strains were suspended in sabouraud’s dextrose broth for 6 hour to give concentration 105 CFU/ml and then inoculated with the culture medium. A total of 8 mm diameter wells were punched into the agar and filled with the sample and solvent blanks (hydro alcohol and hexane). Standard antibiotic, Fluconazole (concentration 1 mg/ml) was used as positive control and fungal plates were incubated at 37oC for 72 h. The diameters of zone of inhibition observed were measured.

 

RESULTS AND DISCUSSION:

Synthesized ZNPs are known in the solution by the color changing from pale green to colorless due to Zn metal ion reduction and from colorless to dark brown color due to capping of stabilizing agent, curcumin. The color change can be easily identified by the naked eye. It was clearly indicates that the formation of well reduced and stabilized ZNPs.

 

UV-Vis spectra studies:

The most convenient and advance technique for characterization of curcumin is UV-Vis spectroscopy. The synthesized turmeric curcumin (CR) was confirmed by the strong broad absorption band observed at around 425 nm. This can be due to either an n→π* transition or a combination of π→π* and n→π* transitions which is shown in Figure 1.

 

Fig. 1UV-Vis spectrum of Curcumin

 

UV-Visible spectroscopy can be used as a simple and reliable method for monitoring the stability of nanoparticle solutions. The absorption spectrum of Zn nanoparticle is shown in Figure 2. It exhibits a strong absorption band at around 315nm which meant the formation of Zn nanoparticles. An excitonic absorption peak is found at about 225nm might be due to the formation of aggregation of Zn nanoparticles which lie much below the band gap wavelength of 315nm. It is also evident that significant sharp absorption of ZNPs indicates the monodispersed nature of the nanoparticle distribution.

 

FT-IR analysis:

FT-IR spectroscopy was used to investigate the interactions between different species and changes in chemical compositions of the mixtures. Figure 3 shows the FT-IR spectrum of curcumin. The important peaks observed from curcumin are 3502 cm-1(Phenolic OH), 1625 cm-1 (C=O), 1520 – 1350 cm-1 (Aromatic C=C, 3 bands), 1272 cm-1 and 1024cm-1(C-O, 2 bands). Figure 4 shows the FT-IR spectrum of curcumin stabilized zinc nanoparticles. From the data obtained, phenolic OH showed its weak broad band in the range of 3389 cm-1 which is assigned to (Ph-OH) group of curcumin moiety. The peak observed at 2926 cm-1 which can be assigned to the -OH stretching of water or ethanol present in the system. The C=O stretching of curcumin at 1625 cm-1 was shifted to a higher wave number at 1704 cm-1 due to interaction with zinc nanoparticles. Three characteristic peaks in the range of 1520 – 1350 cm-1 conforms the aromatic unsaturation (C=C) of stabilized curcumin system. The (C-O) band presence was assigned by the peaks found at 1000-1250 cm-1. The small broad peak obtained at 1030 cm-1 might be due to some oxidation of zinc nanoparticles, i.e., (Zn-O) stretching vibrations.

 

SEM studies

Morphology of synthesized zinc nanoparticles was characterized by SEM analysis. The samples were placed in an evacuated chamber and scanned in a controlled pattern by an electron beam. Interaction of the electron beam with the specimen produces a variety of physical phenomenon that detected, were used to form images and provide information about the specimens. The SEM images of curcumin stabilized zinc nanoparticles are shown in Figure 5. It can be view that the ZNPs formed are well dispersed and evenly distributed in all direction. SEM images of those compounds had shown very clear that most of the particles are cubic shaped morphology of material. The incorporation of low concentrate HCl in the synthesis process helped to avoid agglomeration of nanoparticles.


 

Fig. 2 UV-Vis spectrum of Zn nanoparticles

 

 

Fig. 3FT-IR spectra of curcumin

Fig. 4FT-IR spectra of zinc nanoparticles

 


 

Fig. 5 SEM monographs of ZNPs

 

TEM studies

Figure 6 shows the TEM image of the zinc nanoparticles. These images shows that the particles formed are of nearly spherical morphology. The nanoparticles are moderately dispersed and the average crystallite size of particles in the range of around 23-46 nm.

 

 

Fig. 6TEM monographs of ZNPs

 

From the TEM pictures the aggregations of Zn nanoparticles were observed which confirms the complex formation between curcumin and Zn nanoparticles which reveals the medicinal property of synthesized nanoparticles.

 

Antibacterial activity:

The antibacterial activity of curcumin and curcumin stabilized zinc nanoparticles was evaluated against two gram positive bacteria (S.aureus, B.subtilis), two gram negative bacteria (E.coli, S.bacillus). The compared antibacterial activity resultswith Chloramphenicol are tabulated (Table 1).

 

Fig. 7Antibacterial activity data of complexes

 

 

Table 1Effect of curcumin and zinc nanoparticles on antibacterial activity

Bacterial Species

Zone of inhibition diameter (mm sample-1)

Standard drug (C)

Curcumin (CR)

Zinc nanoparticles (ZNPs)

S. aureus

16

13

18

B.subtilis

18

16

19

E. coli

20

17

16

S.bacillus

21

15

18

 

Figure 7 exhibits the typical antibacterial test results of curcumin and curcumin functionalized zinc nanoparticles obtained by disc diffusion method. It is found that the curcumin stabilized ZNPs have exhibited an appreciable inhibition activity whereas synthesized pure curcumin exhibited moderate inhibition activity only. Zone of inhibition tests showed that ZNPs have a very good antibacterial activity against S. aureus, B. subtilis and S. bacillus strains. Substantially, the zone of inhibition observed by ZNPs towards S. aureus and B. subtilis species has been revealed higher bactericidal activity than standard drug, Chloramphenicol. Therefore, a significant difference is observed from the screening tests that curcumin has  shown low antibacterial activity due to its poor bioavailability and it showed an excellent activity when it is functionalized with ZNPs.

 

 

Antifungal activity:

Curcumin and curcumin stabilized zinc nanoparticles were determined for their antifungal activity against antifungal activity was carried out by agar well diffusion method against four funguses (C. albicans, C. lunata, A. niger, T. simii) and their comparable activity results are tabulated (Table 2).

 

Table 2 Effect of Curcumin and zinc nanoparticles on antifungal activity

Fungal Species

Zone of inhibition diameter (mm sample-1)

Standard drug (C)

Curcumin (CR)

Zinc nanoparticles (ZNPs)

C.albicans

19

16

17

C.lunata

17

14

16

A.niger

20

15

23

T.simii

17

16

17

 

Fig. 8Antifungal activity data of complexes

 

Figure 8 displays the typical antifungal test results of curcumin and curcumin functionalized zinc nanoparticles obtained by agar well diffusion method. The zone of inhibition tests proved that the curcumin stabilized ZNPs has shown considerable antifungal activity against all fungal species tested when compared with synthesized pure curcumin which exhibited a lower inhibition activity only. The obtained results indicated that curcumin stabilized ZNPs have significant inhibition activity against A. niger and T. simii species which was greater than the activity observed by standard drug, Fluconazole. Moreover, ZNPs has shown moderate inhibition activity against C. albicans and C. lunata fungal species. Therefore, curcumin stabilized zinc nanoparticles reveals an attractive antifungal activity over all.

CONCLUSION:

The present work demonstrated a simple and easy synthesis method of curcumin from turmeric plant and preparation of zinc nanoparticles through green route by using lemon extract as a reducer and synthesized curcumin as a stabilizing agent. The experimental results indicated that Zn nanoparticles obtained are in the range of 23-46 nm which are highly stable and appreciable size. The synthesized ZNPs have exhibited an excellent inhibition activity towards S. aureus, B. subtilis bacterial strains and A. niger, T. simii fungal species. Interestingly, the observed inhibition activity was greater than the standard drug tested here. Therefore, the current study clearly provides a promising method to synthesize ZNPs with a natural compound curcumin as a stabilizing agent and prepared Zn nanoparticles are very improved in therapeutic efficacy as antimicrobial agents.

 

ACKNOWLEDGEMENTS:

We gratefully acknowledge Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi for TEM analysis and Nanotechnology Research Centre, SRM University, Chennai for SEM analysis facility. We thank PG and Research Department of Chemistry, V.O. Chidambaram College, Tuticorin for providing IR spectral analysis facility and Department of Chemistry, SFR College for women, Sivakasi for providing UV analysis facility. We also thank AMET University, Chennai, India for their support to do this work.

 

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