Enhanced antibacterial activity of TiO2 nanoparticle surface modified with Garcinia zeylanica extract
© The Author(s) 2017
Received: 26 July 2016
Accepted: 3 January 2017
Published: 12 January 2017
The antibacterial activity of 21 nm TiO2 nanoparticles (NPs) and particles modified with Garcinia zeylanica (G. zeylanica) against Methicillin resistant Staphylococcus aureus was investigated in the presence and absence of light.
Surface modification of TiO2 NPs with the adsorption of G. zeylanica extract, causes to shift the absorption edge of TiO2 NPs to higher wavelength. TiO2 NPs, G. zeylanica pericarp extract showed significant bactericidal activity which was further enhanced in contact with the TiO2 modified G. zeylanica extract.
The antimicrobial activity was enhanced in the presence of TiO2 NPs modified with G. zeylanica and with longer contact time.
Nanotechnology is a nascent technology, gaining popularity globally due to its usefulness in various fields. Nanometals ranging from 1 to 100 nm in size have unique physical and chemical properties which can be exploited for various applications [1, 2]. Further these are promising novel therapeutic agents having antimicrobial and antibiofilm activity.
Development of microbial resistance to antibiotics is a major challenge in the medical field. Therefore, the search for drugs with new modes of action is of major interest in the pharmaceutical and research communities. Two potential sources of novel antimicrobial agents are medicinal plants and nanomaterials [3, 4]. The antimicrobial properties of nanomaterials including metal nanoparticles can be attributed to different mechanisms such as generation of reactive oxygen species, inactivation of cellular enzymes and nucleic acids of the microbes resulting in pore formation in the bacterial cell wall . Among the metal nanoparticles TiO2 NPs are known to be cost effective, stable and safe for humans and the environment. A unique property of TiO2 NPs is the photocatalytic property resulting in enhanced microbicidal activity on exposure to light in the UV range [3, 5]. TiO2 NPs exist in three crystalline phases, where the anastase phase demonstrates high photocatalytic and antimicrobial properties .
Garcinia zeylanica is an endemic plant to Sri Lanka, which belongs to the family Guttiferae (Clusiaceae). Ragunathan et al.  reported antibacterial activity of pericarp of G. zeylanica extract against MRSA, while it had no antimicrobial activity against Candida albicans and Candida parapsilosis . Others have reported antimicrobial activity of Garcinia species against Staphylococcus aureus, Streptococcus pyogenes and some Gram negative bacteria . Garcinia species have many important phytochemicals with antimicrobial potential [9, 10]. The phytochemical analysis of G. zeylanica which is an endemic plant to Sri Lanka, is not yet documented. This study aimed to determine the antibacterial activity of TiO2 NPs modified with G. zeylanica aqueous extract. The combined synergistic effect of phytochemicals and TiO2 NPs were also investigated.
Preparation of Garcinia zeylanica aqueous extract
Dried pericarp of G. zeylanica was collected locally and authenticated at the Bandaranayaka Memorial Ayurveda Research Institute, Navinna, Maharagama, Sri Lanka. The pericarp was rinsed, dried (6 h at 42 °C) and aqueous extract was prepared using 30 g of plant material in 720 ml distilled water, then boiled under low heat to reduce the volume to 120 ml according to Ayurvedic protocol . The plant extract was filtered using sterile Whatman No 1 filter paper. The filtrate was transferred to a sterile glass container and stored in the refrigerator (4 °C) up to 2 weeks.
Characterization and surface modification of TiO2 NPs with G. zeylanica extract
Surface modification of 21 nm TiO2 NPs (Sigma Aldrich) with G. zeylanica aqueous extract was done by refluxing 25 ml of G. zeylanica aqueous extract with 0.30 g of TiO2 (mainly anatase). Solid part was centrifuged and separated. Separated solid was washed with distilled water several times by centrifugation. Washed solid was separated air dried and placed in a vacuum desiccator for 48 h.
Scanning electron microscope (SEM) imaging was performed to understand the surface morphology of TiO2 of the coated petri dishes. SEM imaging was done using FE-SEM (JSM-6320F) at accelerating voltages of 10 kV. Powered X-ray diffraction (XRD) analysis was carried out for the identification of the phase of coated TiO2 using Ultima III (Rigaku) powder diffractometer (Cu-Kα/λ = 0.154 nm). Surface characterization of pure and modified NPs were performed using diffuse reflectance spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Diffuse reflectance spectroscopic studies were carried out using PerkinElmer Lambda 35 spectrophotometer equipped with integrating sphere. ATR-FTIR analysis was carried out using Thermo Scientific Nicolet iS10 FTIR spectrometer.
Phytochemical analysis of the aqueous G. zeylanica extract
Qualitative analysis of various phytocompounds present in the G. zeylanica aqueous extract was done using previously described protocol by Krishnamoorty et al. . Flavanoids, terpenoids, phenols, tannins, cardiac glycosides, carbohydrates, saponins, amino acids, phlobatannin, sterols and alkaloids were detected in this study.
A clinically confirmed isolate of Methicillin resistant S. aureus was obtained from the culture collection at the Department of Microbiology, University of Sri Jayewardenepura. The organism was cultured on Nutrient agar at 37 °C for 18 h. Suspensions of organisms were prepared in sterile normal saline to obtain a 0.5 MacFarland absorbance corresponding to 108 organisms/ml.
Determination of antimicrobial activity of 21 nm TiO2 NPs, and TiO2 NPs modified with G. zeylanica
TiO2 NPs was used at a concentration of 13.9 g/l in sterile miliq (MQ) water . Suspension of TiO2 was prepared by sonication at 35 kHz for 1 h followed by autoclaving for 30 min at 121 °C. The pH of all solutions was adjusted to pH 5.5 prior to coating of the petri dishes.
A separate plate (A) was used as negative control which contained MQ water. Sterile 3 cm petri dishes were coated with (B) TiO2 only, (C) G. zeylanica aqueous extract only and (D) G. zeylanica extract modifies with TiO2. Each petri dish was coated by adding 1 ml of solutions of B, C and D to individual petri dishes. The petri dishes were then evaporated to dryness.
One milliliter of MRSA suspension (108 organisms/ml) was added to each petri dish. The inoculated petri dishes were kept for 1, 4 and 24 h, at room temperature. At the end of each time point 100 μl of suspension was collected from each petri dish and colony forming units/ml (CFU/ml) was determined by spread plate method on Nutrient agar. Further, to determine the enhanced antimicrobial activity due to the photocatalytic activity of TiO2 NPs, one set of petri dishes (tests and control) were incubated for 30 min in sunlight after addition of MRSA suspension and the number of colonies were counted as described above. All experiments were done in triplicates.
Results and discussion
SEM and XRD analysis
Diffuse reflectance, UV–visible and ATR-FTIR study
Phytochemical screening of the aqueous extract of G. zeylanica
Phytochemical screening of the aqueous extract of G. zeylanica
Benzene, 10% NH3
1% aluminium solution
FeCl3, conc. H2SO4
Antibacterial activity of TiO2
The antimicrobial activity of TiO2 even in the absence of photo activation has been well reported . TiO2 carries a positive charge while the surface of microorganisms carry negative charges resulting in an electromagnetic attraction between microorganisms and the TiO2 NPs which leads to oxidation reactions. TiO2 deactivates the cellular enzymes and DNA by coordinating to electron-donating groups, such as: thiols, amides, carbohydrates, indoles, hydroxyls etc. The resulting pits formed in bacterial cell walls lead to increased permeability and cell death .
TiO2 NPs are reported to be non carcinogenic and nontoxic  and are used extensively in food packaging , textile industry , self-cleaning ceramics and glass , in the paper industry for improving the opacity of paper , cosmetic products such as sunscreen creams  etc. Further, TiO2 NPs are used in commercial products such as water purification plants . The antimicrobial activity of TiO2 NPs are exploited in medical devices, in order to prevent biofilm formation and sepsis [35–37].
Antibacterial effect of G. zeylanica aqueous extract
Garcinia zeylanica extracts from other species have been reported to contain hydroxy citric acid, xanthones, flavonoids and benzophenone derivatives such as garcinol . Previous reports have investigated the antimicrobial activity of Garcinia Cambogia , and Garcinia indica .
Antibacterial effect of TiO2 modified with G. zeylanica aqueous extract
When the TiO2 was modified with G. zeylanica extract, there was significant antimicrobial activity in the presence of sunlight (p value = 0.0001) compared to the control. When the modified extract was incubated with MRSA for 1, 4 and 24 h, the antimicrobial activity was seen to be further enhanced with increasing incubation time (p = 0.0002, 0.0007, 0.0044). The percentage reduction of colony counts at all four time points were >99.99%. These results show that the antimicrobial activity of TiO2 was significantly enhanced when modified with G. zeylanica both in the presence and absence of sunlight as shown in Fig. 7. Exposure to sunlight and prolong contact was seen to further enhance the antimicrobial activity.
Comparison of antimicrobial activity of G. zeylanica extract and TiO2 modified with G. zeylanica aqueous extract
G. zeylanica aqueous extract (CFU/ml)
TiO2 modified with G. zeylanica aqueous extract (CFU/ml)
After 30 min sunlight exposure
After 1 h incubation period
After 4 h incubation period
After 24 h incubation period
Anatase 21 nm TiO2 NPs shows antimicrobial activity against MRSA following photoactivation by sunlight. G. zeylanica aqueous extract itself has antimicrobial activity against MRSA. Enhanced antimicrobial activity was observed when the TiO2 was modified with G. zeylanica aqueous extract. Activity against MRSA was further enhanced when TiO2 was modified with G. zeylanica aqueous extract with the exposure to the sunlight.
This work was carried out in collaboration between all authors. Authors SSNF, TDCPG, MMW, HGSPH and PMJ designed the study. Authors ULNHS, NDHA and HDS carried out the experiments and bioassays. All authors contributed to the analysis of results, while authors ULNHS, SSNF, TDCPG, MMW and PMJ wrote the first draft manuscript. All authors read and approved the final manuscript.
The authors would like to thank the National Science Foundation in Sri Lanka for the equipment grant (RG/2013/EQ/07). Appreciation also goes to the University of Sri Jayewardenepura grant (ASP/01/RE/MED/2016/42).
The authors declare that they have no competing interests.
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