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Syntheses and characterization of Syzygium aromaticum, Elettaria cardamomum and Cinnamomum verum modified TiO2 and their biological applications
Materials Science in Semiconductor Processing 105 (2020) 104724
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Syntheses and characterization of Syzygium aromaticum, Elettaria cardamomum and Cinnamomum verum modified TiO2 and their biological applications
T
P. Maheswaria,b, S. Ponnusamyb,∗, S. Harishb,c, C. Muthamizhchelvanb, Y. Hayakawac a
Department of Nautical Science, VELS Institute of Science, Technology & Advanced Studies, Thalambur, 603 103, India Functional Materials and Energy Devices Laboratory, Department of Physics and Nanotechnology, SRM Institute of Science and Technology, Kattankulathur, 603 203, India c Research Institute of Electronics, Shizuoka University, 3-5-1 Johoku, Naka-Ku, Hamamatsu, Shizuoka, 432-8011, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: NPs Bacteria Anticancer Zone of inhibition and antibacterial activities
Diseases form the major roadblock for the living organism. Many chemicals are isolated to cure diseases. But they are found to have side effects due to allergetic chemicals. NPs are found to be good biological agents. But they are also allergetic in nature. In this study, the properties of TiO2 NPs were modified with bio agents such as Clove, Elachi (Cardamom) and Cinnamon. The anticancer and antibacterial properties were also examined. XRD analysis showed the anatase phase for the pure and modified TiO2 samples. The particle sizes were found to be 7.5 nm for pure, 9.5 nm for Clove modified, 9 nm for Elachi modified and 10.5 nm for Cinnamon modified TiO2 NPs. The synthesized TiO2 nanoparticles were tested against bacterial strains such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans using agar diffusion method for antibacterial studies. KB Oral cancer cell line was used to determine the anticancer activities of the synthesized NPs by MTT assay. The synthesized bio modified NPs exhibit significant results against bacterial strains and cancer cell line when compared with the pure NPs.
1. Introduction Nanomaterials are gaining more attraction in day to day life for more than decades. They exhibit significant applications in different fields like biology, chemistry, biotechnology and many more research areas due to their excellent properties [1–4]. Antibacterial and anticancer activities of the NPs are mainly due to their Reactive Oxygen Species (ROS) [5]. Many nanomaterials such as Zn [6,7], Cu [8] and Ag [4] exhibit antibacterial and anticancer activities. Among them TiO2 NPs showed excellent photocatalytic activity [9] and has a tendency to kill cancer cells [10–13]. Jin C et al. [14] observed that the anatse phase of TiO2 was able to generate more ROS than the rutile phase and was experimentally studied using XAFS (X-ray absorption fine structure spectrometry). The biological activities of TiO2 NPs increases with the doped and modified chemical agents. But they are found to be more toxic and expensive. In order to reduce the toxic nature and cost, plant materials can be used as dopants. Plants are found abundant in the environment and they have been used from ancient time in many medicinal systems such as Siddha, Ayurvedha etc. to cure many
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diseases. From the literature, it is reviewed that spices have a tendency to destroy bacterial [15] and cancer cells [16,17]. Among them Clove, Cardamom and Cinnamon are found to exhibit excellent biological activities. Hence modified TiO2 NPs have been prepared using Clove, Cardamom and Cinnamon to enhance the antibacterial and anticancer activities. Cloves are the aromatic flower buds which belong to the Myrtaceae family and the botanical name is Syzygium aromaticum. Chaeib K et al. [18] studied the chemical composition of Clove (Syzygium aromaticum) and observed that eugenol (76.8%), β - caryophyllene (17.4%), α humulene (2.1%), and eugenyl acetate (1.2%) are the main components. Kumar Y et al. [19] observed the antibacterial activity of Clove with two Gram positive (Bacillus cereus, Staphylococcus aureus) and two Gram negative (Salmonella typhi, Escherichia coli) bacterial strains for various concentrations at 2000, 1500 and 1000 ppm using 5 mm disc diameter. Liu H et al. [20] studied the anticancer activity of Syzygium aromaticum using HT - 29 Cells and observed that the ethyl acetate extract of Clove (EAEC) and Oleanolic acid (OA) present in Clove exhibits cytotoxicity against several human cancer cells.
Corresponding author.Department of Physics and Nanotechnology, SRM Institute of Science and Technology Kattankulathur, (PO)-603 203, Tamilnadu, India. E-mail addresses: [email protected] (S. Ponnusamy), [email protected] (S. Harish).
https://doi.org/10.1016/j.mssp.2019.104724 Received 15 April 2019; Received in revised form 16 August 2019; Accepted 4 September 2019 1369-8001/ © 2019 Published by Elsevier Ltd.
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Elachi (Cardamom) is a spice made from the seeds of genera plants Elettaria. The botanical name is Elettaria cardamomum. Korikanthimathm V S et al. [21] observed 1,8-Cineole (36.3%), alpha teripinyl acetate (31.3%) and limonene (11.6%) to be the major constituents of the Cardamom. The basic aroma produced by Cardamom is mainly due to these components. The other components present in cardamom are pinene, sabinene, myrcene, phellandrene, terpinene, cymene, terpinolene, linalool, linalyl acetate, terpineol, citronellol, nerol, geraniol, methyl eugenol and nerlidol. Akrayi H F S [22] studied the antimicrobial activity of Elettaria cardamomum against Staphylococcus aureus and Proteus mirabilis. Majdalawieh Amin F et al. [23] investigated the anticancer and antitumor activity of Cardamom and observed that the aqueous extracts of Cardamom significantly increase the destruction of cancer cells and found that they are natural killer cells. The botanical name of Cinnamon is Cinnamomum verum. It is found in the inner bark of the tree. Meena V [24] pointed out that the major constituents of Cinnamon bark are cinnamaldehyde (65–80%) and eugenol (5–10%). The component Cinnamaldehyde is responsible for the pungent taste and odour.The other compounds present in traces are cinnamic acid, hydroxyl cinnamaldehyde, cinnamyl alcohol, coumarin, cinnamyl acetate, borneol, β-caryophyllene, benzyl benzoate, linalool and eugenyl acetate. Ali N A M et al. [25] studied the chemical composition and antimicrobial activities of Cinnamon bark and leaf extracts and concluded that the Cinnamon bark has efficient antibacterial activity than leaf extracts due to their major components present in the bark. Wang H M et al. [26] showed the cytotoxic nature of Cinnamon against human skin cancer melanoma A375.S2 cells. Fig. 1 represents the chemical structure of eugenol, oleanolic acid, 1,8-cineole and cinnamaldehyde. Titanium dioxide NPs are synthesized by various methods such as sol gel [27], coprecipitation [28], hydrothermal [29,30], spray pyrolysis [31], DC reactive magnetron sputtering [32]. Tam K H et al. [33] suggested that hydrothermal methods are gaining more interest because of their low cost and environmentally friendly method. In the present work, Pure, Clove modified, Elachi modified and Cinnamon modified TiO2 were synthesized by hydrothermal technique. They were subjected to characterizations such as XRD (X-Ray diffraction), UV–vis (UV–vis Spectrophotometer), FTIR (Fourier Transform InfraRed spectrometer), TEM (Transmission Electron Microscopy), MTT assay and Agar diffusion technique. The modified samples exhibit
Fig. 2. XRD patterns of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples.
Fig. 3. UV Spectra of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples.
better activity than the unmodified samples. 2. Experimental methods Titanium tetraisopropoxide and isopropyl alcohol were purchased from Sigma - Aldrich Chemicals. Dried Clove buds, Cardamom and Cinnamon were purchased from local Supermarket. 2.1. Preparation of TiO2 NPs 5 mL of Titanium (IV) tetraisopropoxide, 10 mL of isopropyl alcohol and 70 mL of deionised water were mixed separately and then stirred for about 2 h at room temperature. The sol then obtained due to stirring was transferred to the Teflon autoclave and then subjected to heating for about 2 h at 200 °C. The solution was centrifuged and then rinsed with water and ethanol to get rid of impurities and then kept in oven at 100 °C. The dried powder was annealed at 350 °C for removal of impurities. The final product was named as [T1].
Fig. 1. Chemical structure of Eugenol, Oleanolic acid, 1,8-Cineole and Cinnamaldehyde. 2
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Fig. 4. TEM images of (a) Clove modified TiO2 (b) Elachi modified TiO2 (c) Cinnamon modified TiO2 (d) Clove - Elachi modified TiO2 (e) Elachi - Cinnamon modified TiO2 (f) Cinnamon - Clove modified TiO2.
Fig. 5. HRTEM images of (a) Clove modified TiO2 (b) Elachi modified TiO2 (c) Cinnamon modified TiO2 (d) Clove - Elachi modified TiO2 (e) Elachi - Cinnamon modified TiO2 (f) Cinnamon - Clove modified TiO2.
- Elachi, Elachi - Cinnamon, Clove - Cinnamon extracts and finally Elachi - TiO2 [T3], Cinnamon - TiO2 [T4], Clove - Elachi modified TiO2 [T5], Elachi - Cinnamon modified TiO2 [T6] and Clove - Cinnamon modified TiO2 [T7] NPs respectively were obtained. In the case of Clove - Elachi extract, 2 mL of Clove and 2 mL of Elachi was used for the synthesis process.
2.2. Preparation of extract and modified TiO2 samples Clove was washed with deionised water three times to remove impurities. 10 g of clove was trampled and then heated with 100 mL of deionised water at 60 °C for about 30 min. Then it was filtered using Whatman filter paper, and the extract was protected at 4 °C for further use as Clove extract. Similarly, using Cardamom (Elachi) and Cinnamon, the extracts were prepared as Elachi and Cinnamon extracts. About 0.2 g of synthesized Titanium dioxide powder was mixed with 20 mL of deionised water. To the above mixture, 4 mL of Clove extract was poured and stirred at room temperature thoroughly for 2 h and then subjected to centrifuging process, which was finally dried in oven at 100 °C. Thus, the Clove modified TiO2 NPs [T2] were obtained. Similar method was repeated by using 4 mL of Elachi, Cinnamon, Clove
2.3. Characterization of the pure and modified TiO2 samples For the phase analysis and characterization, XPERT - PRO diffractometer was used with λ = 1.5406 Ả. The particle size of the samples were investigated by transmission electron microscopy (TEM, Model JEOL 3010, 300 kV). UV Spectral studies were carried out using a UV–Vis–NIR Spectrophotometer. Surface functional groups were 3
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Fig. 6. Bar distribution of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples.
determined using PerkinElmer Infrared spectrometer. 2.3.1. Antibacterial technique The antibacterial activities of T1, T2, T3, T4, T5, T6 and T7 samples were investigated by Agar well diffusion technique. Escherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 530), Pseudomonas aeruginosa (MTCC 1688), Staphylococcus aureus (MTCC 737) and Streptococcus mutans (MTCC 890) were acquired from Microbial Type culture collection and gene bank (MTCC) at Chandigarh. The bacterial strains were maintained on nutrient agar. Then it was subjected to Agar diffusion test using streptomycin as a standard control and the inhibition zone was measured. 2.3.2. Cell viability technique In the 96-well plate the cells were seeded and then the plate was maintained at 37 °C for 24 h. After one day, the plates were removed from the incubator and then the MTT reagent was added to it. In order to escape from light, the plate was covered with aluminum foil. It was then incubated for 3 h and then after the removal of the reagent, 100 μl of DMSO was added to this process and mixed well. The readings were recorded by using 570 and 630 nm as reference wavelength. The optical density was calculated for the viable cells using the
Fig. 7. FTIR spectra of (a) T1 (Pure TiO2).
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Fig. 8. FTIR Spectra of (a) clove extract (b) elachi extract (c) cinnamon extract (d) clove - elachi extract (e) elachi - cinnamon extract and (f) cinnamon - clove extract.
technique and the size of the synthesized particle was found to be 7 nm. According to Wei X et al. [36], the phase obtained during the sol-hydrothermal technique was found to be anatase in nature. A small peak at 30.7° observed in the XRD pattern of the sample T1 is assigned to (2 1 1) plane of the brookite phase [37]. After the addition of plant extracts, for the samples T2, T3, T4, T5, T6 and T7, the peak intensity of (2 1 1) plane decreases as compared to the pure sample (T1). The XRD results clearly confirms the incorporation of plant extracts on the TiO2.
given formula:
% of cell viability =
(Absorbance O. D by sample ) x 100 (Absorbance O. D by control)
3. Results and discussion 3.1. X-ray diffraction analyses Fig. 2 depicted the X-ray patterns for T1, T2, T3, T4, T5, T6 and T7 NPs. The peaks observed at 25.3°, 37.8°, 48°, 54.7°, 63°, 70° and 75.7° denotes the crystal planes of (1 0 1), (0 0 4), (2 0 0), (1 0 5), (2 0 4), (2 2 0) and (2 1 5), respectively, which is in good agreement with the JCPDS file No: 21–1272 of anatase TiO2 [34]. Nainani R et al. [35] have synthesized silver doped titanium dioxide NPs by photodeposition
3.2. UV spectra analyses The absorbance spectra for the synthesized pure and modified NPs are observed using UV–Vis Spectrophotometer. Fig. 3 showed the UV spectra for pure and bio modified TiO2. The maximum absorbance for the Pure TiO2 is observed at 264 nm [T1] which is in good agreement 5
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assigned for Pure TiO2 NPs. The absorption peaks of the samples T2, T3, T4, T5, T6 and T7 are 313, 304, 330, 260, 307 and 268 nm, respectively. For the bio-modified TiO2 samples, the absorbance peaks are slightly red shifted due to the chemisorption of Elachi, Cinnamon, Elachi - Cinnamon and Clove - Cinnamon molecules on the TiO2 surface. But in the case of Clove - Elachi modified TiO2 sample [T5] shows blue shift in comparison with the pure TiO2. A similar result was observed by Gharibshahi E et al. [40] in his studies reported that the size of the particle decreases with respect to the blue shift. A shift in the peak showed that there is a reduction in the particle size which is in good agreement with the TEM results. 3.3. TEM and HRTEM analyses Fig. 4 and 5 showed the TEM and HRTEM images of the pure and bio modified samples. It was observed that the spherical shaped TiO2 nanoparticles were formed in the synthesized samples. The size of the nanoparticle varies with different sizes depending on the bio modified samples (Clove, Elachi, Cinnamon, Clove - Elachi, Elachi - Cinnamon and Cinnamon – Clove molecules). The size distributions were determined from TEM images by considering the diameter for spherical shaped nanoparticles. The average particle size of the pure, and bio modified TiO2 NPs samples are shown in Fig. 6 as a histogram. The average particle size of sample T1 is 7.5 nm, T2 is 9.5 nm, T3, is 9 nm T4 is 10.5 nm, T5 is 6.5 nm, T6 is 9 nm and T7 is 7.5 nm, respectively. In these complex biological fluids, nanoparticles can form large aggregates due to changes in pH, ionic strength, and the presence of biomolecules that disturb the delicate balance. Fig. 5 shows the HRTEM images of the Pure and modified NPs. Crystalline fringes in the HRTEM images shows
Fig. 9. FTIR spectra of (a) T2 (b) T3 (c) T4 (d) T5 (e) T6 and (f) T7.
with Rathod P B et al. [38]. He reported the maximum absorbance of titanium dioxide NPs are found to lie in between 220 and 320 nm. Hasan et al. [39] pointed out that the absorbance at 337 nm could be
Fig. 10. Toxicity profiles (concentration vs cell viability) of the KB cells treated with T1, T2, T3 T4, T5, T6 and T7 samples for various concentrations [Cells were treated with different concentrations of the samples for 24 h. At the end of the incubation period, cell viability was determined by MTT assay]. 6
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Fig. 11. Inverted Microscope images of the KB oral cancer cells treated with 10 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
Fig. 12. Inverted Microscope images of the KB oral cancer cells treated with 20 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
C]C stretching vibrations respectively. Fig. 7 shows the broad peak at 633 cm−1 that denotes the O–Ti–O vibration bands [44]. The peaks at 1633 and 3420 cm−1 are the characteristic vibrations of hydroxyl groups. From Fig. 9, it is observed that there is a slight shift at 1635 cm−1 for Clove modified NPs when compared with 1633 cm−1 of Pure NPs. This confirms that there is a chemical reaction between the eugenol in Clove and titanium dioxide nanoparticles. In a similar way, a peak is observed at 1636 cm−1 for Elachi and Cinnamon respectively.
the good crystalline nature of the TiO2 samples. 3.4. FTIR spectra analyses Figs. 7–9 shows the FTIR spectral analyses of pure TiO2, extracts (Clove, Elachi, Cinnamon, Clove - Elachi, Elachi - Cinnamon, Cinnamon - Clove) and bio modified TiO2 NPs [T2, T3, T4, T5, T6 and T7]. From Fig. 8a, the key component of Clove, eugenol shows the characteristic band at 1607 cm−1 due to the C]C stretching vibrations [41]. In the case of Elachi and Cinnamon, the main constituents are 1,8-cineole and cinnamaldehyde. The characteristic peak of cineole [42] and cinnamaldehyde [43] occurs at 1619 cm−1 and 1616 cm−1 which represent
3.5. Anticancer activities of pure and modified samples Fig. 10 shows the anticancer effect of pure, Clove, Elachi, 7
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Fig. 13. Inverted Microscope images of the KB oral cancer cells treated with 30 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
Fig. 14. Inverted Microscope images of the KB oral cancer cells treated with 40 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
50.67%, 20.30%, 4.60%, 6.77%, 14.65%, 13.62% and 21.25% viabilities respectively. Among the modified samples T3 shows excellent anticancer activities when compared with pure and other modified samples. There is a decrease in the band gap of the T3 sample which is responsible for the production of superoxide radicals. This shows that T3 sample releases a greater number of free radicals which is responsible for Reactive Oxygen species (ROS) generation. This ROS is responsible for the destruction of the cancer cells. The component 1,8cineole present in Elachi may increase the anticancer nature in comparison with the pure Titanium dioxide NPs. The anticancer activity increases with the increase in the concentration of the sample. This can be explained through the phenomena that when the concentration increases more number of the NPs penetrate the cells easily and produce more number of superoxide radicals
Cinnamon, Clove - Elachi, Elachi - Cinnamon and Cinnamon - Clove modified TiO2 NPs against KB oral cancer cell line. Five different concentrations are used to analyze the anticancerous nature of the samples. Camptothecin is the standard drug used for the analysis. It is observed that variable cell viability is seen for various samples with same concentration and the bio modified samples enhance the anticancerous nature when modified with the pure sample. From the Figures, it is observed that for 10 μg/mL of T1, T2, T3, T4, T5, T6 and T7 samples showed 98.28%, 81.91%, 44.53%, 31.34%, 70.86%, 55.69% and 55.18% viabilities, for 20 μg/mL showed 77.32%, 65.89%, 23.75%, 18.78%, 49.01%, 42.41% and 43.70% viabilities, for 30 μg/mL showed 69.73%, 45.41%, 12.82%, 15.71%, 32.04%, 25.10% and 35.38% viabilities, for 40 μg/mL showed 60.07%, 35.13%, 5.51%, 11.83%, 21.33%, 20.13% and 28.19% viabilities and for 50 μg/mL showed 8
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Fig. 15. Inverted Microscope images of the KB oral cancer cells treated with 50 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
Fig. 16. Zone of Inhibition in mm of (a) T1 [T24] (b) T2 [ T49] (c) T3 [ T50] (d) T4 [ T51] (e) T5 [T52] (f) T6 [T53] and (g) T7 [T 54] samples against Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus & Streptococcus mutans.
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Fig. 17. Zone of Inhibition in mm of (a) T1 [T24] (b) T2 [ T49] (c) T3 [ T50] (d) T4 [ T51] (e) T5 [T52] (f) T6 [T52] and (g) T7 [T 54] samples against Staphylococcus aureus & Streptococcus mutans.
Syzygium aromaticum is the best anticancer agent for lung cancer. He also pointed out that the eugenol; key component of Clove is acting as a good agent for production of oxide radicals. The cinnamaldehyde present in the Cinnamon can be responsible for the damage of the cancer cells. Meena V et al. [24] studied the anticancerous nature of the cinnamaldehyde from Cinnamon bark against Breast cancer cell line (MCF7). She observed that at 200 μg/mL concentration of the sample damaged 32.3% cancer cells. Figs. 11–15 represents the microscopic images of Oral cancer cells with modified plant NPs for different concentrations.
which is responsible for the damage of the cancer cells [45]. Murata S et al. [46] discussed the antitumor effect of 1,8-cineole against colon cancer. He observed that eucalyptol (1,8-cineole) exhibit antitumor activity against HCT116 and RKO (human colon cancer) cell lines when subjected to Western blot analysis and cell viability assay. Moteki H [47] studied the use of cineole on the growth of Molt 4B, HL-60 and KATO III cell lines. He used 5, 7.5 and 10 μm concentration of the cineole and subjected to the cell line for 72 h and then the viability is evaluated using Trypan blue exclusion method. He observed that 1,8cineole exert activity against human leukemia cells but no activity is seen in the human stomach cancer cell line (KATO - III). Ashour H M [48] studied the anti tumor properties of essential oils of Eucalyptus species on MCF7 (Human breast adenocarcinoma) and HEPG2 (Human hepatocellular carcinoma) cell lines. He observed that E.torquata leaves exhibit cytotoxic activity against MCF7 cell lines. The presence of eugenol in the Clove may be responsible for the anticancer nature of the Clove modified TiO2. Dwivedi V et al. [49] studied the anticancer nature of the water, ethyl alcohol and oil extracts of Clove. He showed that maximum death occurs in TE-13, esophageal cancer cell line due to apoptosis. He pointed out that the cytotoxic activities of the cells are due to the presence of eugenol in the Clove extract. This eugenol is responsible for all pharmacological activities and also kills maximum cancer cells. Banerjee S [50] observed that the
3.6. Antibacterial activities of pure and modified samples Fig. 16 and Fig. 17 showed the zone of inhibition for T1, T2, T3, T4, T5, T6 and T7 samples against Gram-positive bacteria such as Staphylococcus aureus, Streptococcus mutans and Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. It is observed that zone of inhibition is maximum for Clove modified [T2] TiO2 NPs against all bacterial Strains. No activity is seen for T4 and T7 samples for any bacterial strains. Maximum zone of Inhibition for T1, T2 and T6 is observed for Gram positive bacteria such as Streptococcus mutans. Similarly, for T3 and T5 samples maximum zone is seen against Pseudomonas aeruginosa. It is seen that eugenol 10
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Fig. 18. Graph for antibacterial activities of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples against Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus & Streptococcus mutans.
reported about the elements present and their pharmacological activities of the Syzygium aromaticum essential oil extracted from the dried flower buds of clove. He observed eugenol to be one of the major chemical constituents. The oil is subjected to Agar disk diffusion method for antibacterial activity and Gentamicin is used as a standard antibiotic and zone of inhibition is measured by using bacterial strains. The maximum zone is observed against S. epidermidis and this maximum activity may be due to the presence of eugenol in the sample. Kumar Y [19] compared the antibacterial nature of the Clove and Garlic extracts. He also pointed that Clove showed 23, 15 and 10 mm inhibition zone against Salmonella typhi at 2000, 1500 and 1000 ppm at concentration. The T1 NPs produces more amount of reactive oxygen species and they easily enter inside the antibacterial activities when compared with other pure and modified sample. The biomolecules present in the Clove modified TiO2 enhances the antibacterial activity of the samples. Fig. 19 represents graphical representation of biological applications of the samples.
present in the Clove may be responsible for the biological activities, Fig. 18 represents the Graphical representation of antibacterial activities of pure and spices modified samples against Gram positive and Gram negative bacterial organisms. Burt S [51] in his review article pointed out that eugenol, present in the Clove is responsible for the breaking up of the cells which in turn damages the cell wall of the Bacillus cereus. Sharma A et al. [52] studied the antimicrobial nature of the aqueous Clove and Ginger extracts against Streptococcus mutans. He observed that 5% of Clove extract shows no activity whereas 50% of extract showed excellent activity against S. mutans. He studied the extracts of Clove and Ginger for antimicrobial nature and found that Clove extracts showed more antibacterial nature when compared with that of the Ginger extracts. Xu J G et al. [53] did his research with essential oil of Clove for the determination of its antibacterial nature. He reported that eugenol forms the major constituent of the Clove (72.3%) and Clove is found to destroy the bacterial cell wall and membrane. At the same time, it stops the normal procedure for the activation of DNA and proteins that are important for the growth of bacteria. Liu Q et al. [15] studied about the antibacterial and antifungal activity of the various spices such as Syzygium aromaticum, Origanum vulgare, Thymus vulgaris, Cinnamomum verum and Cuminum cyminum against food spoilage pathogens like Bacillus subtilis and Pseudomonas fluorescens and Staphylococcus aureus. He concluded that spices can become new and safer antimicrobial agents. Chaeib K et al. [18]
4. Conclusion The Syzygium aromaticum, Elettaria cardamomum and Cinnamomum verum modified NPs were synthesized using hydrothermal method and characterized using XRD, UV, FTIR and TEM analysis. The research experiments analysed the biological activities of the synthesized samples which confirmed that the modifiers have enhanced the biological 11
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Fig. 19. Graphical representation of Biological applications.
activities of the TiO2 NPs. The inhibition bacterial zones are found to be maximum for Syzygium aromaticum modified TiO2 samples in comparison with the pure samples against Gram positive and Gram negative bacterial strains. The Elachi modified TiO2 NPs showed minimum cell viability among the cancer cells at 50 μg/mL concentration of the sample. Hence from the research findings, it is concluded that modified samples are found to be an excellent anticancer and antibacterial agent when compared with the pure samples.
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Acknowledgements The authors are thankful to the Research Institute of Electronics, Shizuoka University, Japan for their TEM analysis. Authors extend their regards to the Nanotechnology Research Center, SRM Institute of Science and Technology for XRD measurement. Authors are grateful to Research Institute, Biotechnology Department, ISISM, SRM Institute of Science and Technology for their valuable facilities. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mssp.2019.104724. References [1] M. Liong, B. France, K.A. Bradley, J.I. Zink, Adv. Mater. 21 (2009) 1684–1689. [2] V.E. Copcia, C. Luchian, S. Dunca, N. Bilba, C.M. Hristodor, J. Mater. Sci. 46 (2011) 7121–7128. [3] G. Ren, D. Hu, E.W.C. Cheng, M.A. Vargas -Reus, P. Reip, R.P. Allaker, Int. J. Antimicrob. Agents 33 (2009) 587–590. [4] J.P. Ruparelia, A.K. Chatteriee, S.P. Duttagupta, S. Mukherji, Acta Biomater. 4 (2008) 707–716. [5] S. Parham, D.H.B. Wicaksono, S. Bagherbaigi, S.L. Lee, H. Nur, J. Chin. Chem. Soc. 63 (2016) 385–393. [6] C. Bergs, L. Brück, R. Ruben, Rosencrantz, G. Conrads, L. Elling, A. Pich, RSC Adv. 7 (2017) 38998–39010. [7] R. Borda d’ Água, R. Branquinho, M.P. Duarte, E. Maurício, A.L. Fernando, R. Martins, E. Fortunato, New J. Chem. 42 (2018) 1052–1060. [8] K.Y. Yoon, J.H. Byeon, J.H. Park, J. Hwang, Sci. Total Environ. 373 (2007) 572–575.
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Syntheses and characterization of Syzygium aromaticum, Elettaria cardamomum and Cinnamomum verum modified TiO2 and their biological applications
T
P. Maheswaria,b, S. Ponnusamyb,∗, S. Harishb,c, C. Muthamizhchelvanb, Y. Hayakawac a
Department of Nautical Science, VELS Institute of Science, Technology & Advanced Studies, Thalambur, 603 103, India Functional Materials and Energy Devices Laboratory, Department of Physics and Nanotechnology, SRM Institute of Science and Technology, Kattankulathur, 603 203, India c Research Institute of Electronics, Shizuoka University, 3-5-1 Johoku, Naka-Ku, Hamamatsu, Shizuoka, 432-8011, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: NPs Bacteria Anticancer Zone of inhibition and antibacterial activities
Diseases form the major roadblock for the living organism. Many chemicals are isolated to cure diseases. But they are found to have side effects due to allergetic chemicals. NPs are found to be good biological agents. But they are also allergetic in nature. In this study, the properties of TiO2 NPs were modified with bio agents such as Clove, Elachi (Cardamom) and Cinnamon. The anticancer and antibacterial properties were also examined. XRD analysis showed the anatase phase for the pure and modified TiO2 samples. The particle sizes were found to be 7.5 nm for pure, 9.5 nm for Clove modified, 9 nm for Elachi modified and 10.5 nm for Cinnamon modified TiO2 NPs. The synthesized TiO2 nanoparticles were tested against bacterial strains such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans using agar diffusion method for antibacterial studies. KB Oral cancer cell line was used to determine the anticancer activities of the synthesized NPs by MTT assay. The synthesized bio modified NPs exhibit significant results against bacterial strains and cancer cell line when compared with the pure NPs.
1. Introduction Nanomaterials are gaining more attraction in day to day life for more than decades. They exhibit significant applications in different fields like biology, chemistry, biotechnology and many more research areas due to their excellent properties [1–4]. Antibacterial and anticancer activities of the NPs are mainly due to their Reactive Oxygen Species (ROS) [5]. Many nanomaterials such as Zn [6,7], Cu [8] and Ag [4] exhibit antibacterial and anticancer activities. Among them TiO2 NPs showed excellent photocatalytic activity [9] and has a tendency to kill cancer cells [10–13]. Jin C et al. [14] observed that the anatse phase of TiO2 was able to generate more ROS than the rutile phase and was experimentally studied using XAFS (X-ray absorption fine structure spectrometry). The biological activities of TiO2 NPs increases with the doped and modified chemical agents. But they are found to be more toxic and expensive. In order to reduce the toxic nature and cost, plant materials can be used as dopants. Plants are found abundant in the environment and they have been used from ancient time in many medicinal systems such as Siddha, Ayurvedha etc. to cure many
∗
diseases. From the literature, it is reviewed that spices have a tendency to destroy bacterial [15] and cancer cells [16,17]. Among them Clove, Cardamom and Cinnamon are found to exhibit excellent biological activities. Hence modified TiO2 NPs have been prepared using Clove, Cardamom and Cinnamon to enhance the antibacterial and anticancer activities. Cloves are the aromatic flower buds which belong to the Myrtaceae family and the botanical name is Syzygium aromaticum. Chaeib K et al. [18] studied the chemical composition of Clove (Syzygium aromaticum) and observed that eugenol (76.8%), β - caryophyllene (17.4%), α humulene (2.1%), and eugenyl acetate (1.2%) are the main components. Kumar Y et al. [19] observed the antibacterial activity of Clove with two Gram positive (Bacillus cereus, Staphylococcus aureus) and two Gram negative (Salmonella typhi, Escherichia coli) bacterial strains for various concentrations at 2000, 1500 and 1000 ppm using 5 mm disc diameter. Liu H et al. [20] studied the anticancer activity of Syzygium aromaticum using HT - 29 Cells and observed that the ethyl acetate extract of Clove (EAEC) and Oleanolic acid (OA) present in Clove exhibits cytotoxicity against several human cancer cells.
Corresponding author.Department of Physics and Nanotechnology, SRM Institute of Science and Technology Kattankulathur, (PO)-603 203, Tamilnadu, India. E-mail addresses: [email protected] (S. Ponnusamy), [email protected] (S. Harish).
https://doi.org/10.1016/j.mssp.2019.104724 Received 15 April 2019; Received in revised form 16 August 2019; Accepted 4 September 2019 1369-8001/ © 2019 Published by Elsevier Ltd.
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Elachi (Cardamom) is a spice made from the seeds of genera plants Elettaria. The botanical name is Elettaria cardamomum. Korikanthimathm V S et al. [21] observed 1,8-Cineole (36.3%), alpha teripinyl acetate (31.3%) and limonene (11.6%) to be the major constituents of the Cardamom. The basic aroma produced by Cardamom is mainly due to these components. The other components present in cardamom are pinene, sabinene, myrcene, phellandrene, terpinene, cymene, terpinolene, linalool, linalyl acetate, terpineol, citronellol, nerol, geraniol, methyl eugenol and nerlidol. Akrayi H F S [22] studied the antimicrobial activity of Elettaria cardamomum against Staphylococcus aureus and Proteus mirabilis. Majdalawieh Amin F et al. [23] investigated the anticancer and antitumor activity of Cardamom and observed that the aqueous extracts of Cardamom significantly increase the destruction of cancer cells and found that they are natural killer cells. The botanical name of Cinnamon is Cinnamomum verum. It is found in the inner bark of the tree. Meena V [24] pointed out that the major constituents of Cinnamon bark are cinnamaldehyde (65–80%) and eugenol (5–10%). The component Cinnamaldehyde is responsible for the pungent taste and odour.The other compounds present in traces are cinnamic acid, hydroxyl cinnamaldehyde, cinnamyl alcohol, coumarin, cinnamyl acetate, borneol, β-caryophyllene, benzyl benzoate, linalool and eugenyl acetate. Ali N A M et al. [25] studied the chemical composition and antimicrobial activities of Cinnamon bark and leaf extracts and concluded that the Cinnamon bark has efficient antibacterial activity than leaf extracts due to their major components present in the bark. Wang H M et al. [26] showed the cytotoxic nature of Cinnamon against human skin cancer melanoma A375.S2 cells. Fig. 1 represents the chemical structure of eugenol, oleanolic acid, 1,8-cineole and cinnamaldehyde. Titanium dioxide NPs are synthesized by various methods such as sol gel [27], coprecipitation [28], hydrothermal [29,30], spray pyrolysis [31], DC reactive magnetron sputtering [32]. Tam K H et al. [33] suggested that hydrothermal methods are gaining more interest because of their low cost and environmentally friendly method. In the present work, Pure, Clove modified, Elachi modified and Cinnamon modified TiO2 were synthesized by hydrothermal technique. They were subjected to characterizations such as XRD (X-Ray diffraction), UV–vis (UV–vis Spectrophotometer), FTIR (Fourier Transform InfraRed spectrometer), TEM (Transmission Electron Microscopy), MTT assay and Agar diffusion technique. The modified samples exhibit
Fig. 2. XRD patterns of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples.
Fig. 3. UV Spectra of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples.
better activity than the unmodified samples. 2. Experimental methods Titanium tetraisopropoxide and isopropyl alcohol were purchased from Sigma - Aldrich Chemicals. Dried Clove buds, Cardamom and Cinnamon were purchased from local Supermarket. 2.1. Preparation of TiO2 NPs 5 mL of Titanium (IV) tetraisopropoxide, 10 mL of isopropyl alcohol and 70 mL of deionised water were mixed separately and then stirred for about 2 h at room temperature. The sol then obtained due to stirring was transferred to the Teflon autoclave and then subjected to heating for about 2 h at 200 °C. The solution was centrifuged and then rinsed with water and ethanol to get rid of impurities and then kept in oven at 100 °C. The dried powder was annealed at 350 °C for removal of impurities. The final product was named as [T1].
Fig. 1. Chemical structure of Eugenol, Oleanolic acid, 1,8-Cineole and Cinnamaldehyde. 2
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Fig. 4. TEM images of (a) Clove modified TiO2 (b) Elachi modified TiO2 (c) Cinnamon modified TiO2 (d) Clove - Elachi modified TiO2 (e) Elachi - Cinnamon modified TiO2 (f) Cinnamon - Clove modified TiO2.
Fig. 5. HRTEM images of (a) Clove modified TiO2 (b) Elachi modified TiO2 (c) Cinnamon modified TiO2 (d) Clove - Elachi modified TiO2 (e) Elachi - Cinnamon modified TiO2 (f) Cinnamon - Clove modified TiO2.
- Elachi, Elachi - Cinnamon, Clove - Cinnamon extracts and finally Elachi - TiO2 [T3], Cinnamon - TiO2 [T4], Clove - Elachi modified TiO2 [T5], Elachi - Cinnamon modified TiO2 [T6] and Clove - Cinnamon modified TiO2 [T7] NPs respectively were obtained. In the case of Clove - Elachi extract, 2 mL of Clove and 2 mL of Elachi was used for the synthesis process.
2.2. Preparation of extract and modified TiO2 samples Clove was washed with deionised water three times to remove impurities. 10 g of clove was trampled and then heated with 100 mL of deionised water at 60 °C for about 30 min. Then it was filtered using Whatman filter paper, and the extract was protected at 4 °C for further use as Clove extract. Similarly, using Cardamom (Elachi) and Cinnamon, the extracts were prepared as Elachi and Cinnamon extracts. About 0.2 g of synthesized Titanium dioxide powder was mixed with 20 mL of deionised water. To the above mixture, 4 mL of Clove extract was poured and stirred at room temperature thoroughly for 2 h and then subjected to centrifuging process, which was finally dried in oven at 100 °C. Thus, the Clove modified TiO2 NPs [T2] were obtained. Similar method was repeated by using 4 mL of Elachi, Cinnamon, Clove
2.3. Characterization of the pure and modified TiO2 samples For the phase analysis and characterization, XPERT - PRO diffractometer was used with λ = 1.5406 Ả. The particle size of the samples were investigated by transmission electron microscopy (TEM, Model JEOL 3010, 300 kV). UV Spectral studies were carried out using a UV–Vis–NIR Spectrophotometer. Surface functional groups were 3
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Fig. 6. Bar distribution of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples.
determined using PerkinElmer Infrared spectrometer. 2.3.1. Antibacterial technique The antibacterial activities of T1, T2, T3, T4, T5, T6 and T7 samples were investigated by Agar well diffusion technique. Escherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 530), Pseudomonas aeruginosa (MTCC 1688), Staphylococcus aureus (MTCC 737) and Streptococcus mutans (MTCC 890) were acquired from Microbial Type culture collection and gene bank (MTCC) at Chandigarh. The bacterial strains were maintained on nutrient agar. Then it was subjected to Agar diffusion test using streptomycin as a standard control and the inhibition zone was measured. 2.3.2. Cell viability technique In the 96-well plate the cells were seeded and then the plate was maintained at 37 °C for 24 h. After one day, the plates were removed from the incubator and then the MTT reagent was added to it. In order to escape from light, the plate was covered with aluminum foil. It was then incubated for 3 h and then after the removal of the reagent, 100 μl of DMSO was added to this process and mixed well. The readings were recorded by using 570 and 630 nm as reference wavelength. The optical density was calculated for the viable cells using the
Fig. 7. FTIR spectra of (a) T1 (Pure TiO2).
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Fig. 8. FTIR Spectra of (a) clove extract (b) elachi extract (c) cinnamon extract (d) clove - elachi extract (e) elachi - cinnamon extract and (f) cinnamon - clove extract.
technique and the size of the synthesized particle was found to be 7 nm. According to Wei X et al. [36], the phase obtained during the sol-hydrothermal technique was found to be anatase in nature. A small peak at 30.7° observed in the XRD pattern of the sample T1 is assigned to (2 1 1) plane of the brookite phase [37]. After the addition of plant extracts, for the samples T2, T3, T4, T5, T6 and T7, the peak intensity of (2 1 1) plane decreases as compared to the pure sample (T1). The XRD results clearly confirms the incorporation of plant extracts on the TiO2.
given formula:
% of cell viability =
(Absorbance O. D by sample ) x 100 (Absorbance O. D by control)
3. Results and discussion 3.1. X-ray diffraction analyses Fig. 2 depicted the X-ray patterns for T1, T2, T3, T4, T5, T6 and T7 NPs. The peaks observed at 25.3°, 37.8°, 48°, 54.7°, 63°, 70° and 75.7° denotes the crystal planes of (1 0 1), (0 0 4), (2 0 0), (1 0 5), (2 0 4), (2 2 0) and (2 1 5), respectively, which is in good agreement with the JCPDS file No: 21–1272 of anatase TiO2 [34]. Nainani R et al. [35] have synthesized silver doped titanium dioxide NPs by photodeposition
3.2. UV spectra analyses The absorbance spectra for the synthesized pure and modified NPs are observed using UV–Vis Spectrophotometer. Fig. 3 showed the UV spectra for pure and bio modified TiO2. The maximum absorbance for the Pure TiO2 is observed at 264 nm [T1] which is in good agreement 5
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assigned for Pure TiO2 NPs. The absorption peaks of the samples T2, T3, T4, T5, T6 and T7 are 313, 304, 330, 260, 307 and 268 nm, respectively. For the bio-modified TiO2 samples, the absorbance peaks are slightly red shifted due to the chemisorption of Elachi, Cinnamon, Elachi - Cinnamon and Clove - Cinnamon molecules on the TiO2 surface. But in the case of Clove - Elachi modified TiO2 sample [T5] shows blue shift in comparison with the pure TiO2. A similar result was observed by Gharibshahi E et al. [40] in his studies reported that the size of the particle decreases with respect to the blue shift. A shift in the peak showed that there is a reduction in the particle size which is in good agreement with the TEM results. 3.3. TEM and HRTEM analyses Fig. 4 and 5 showed the TEM and HRTEM images of the pure and bio modified samples. It was observed that the spherical shaped TiO2 nanoparticles were formed in the synthesized samples. The size of the nanoparticle varies with different sizes depending on the bio modified samples (Clove, Elachi, Cinnamon, Clove - Elachi, Elachi - Cinnamon and Cinnamon – Clove molecules). The size distributions were determined from TEM images by considering the diameter for spherical shaped nanoparticles. The average particle size of the pure, and bio modified TiO2 NPs samples are shown in Fig. 6 as a histogram. The average particle size of sample T1 is 7.5 nm, T2 is 9.5 nm, T3, is 9 nm T4 is 10.5 nm, T5 is 6.5 nm, T6 is 9 nm and T7 is 7.5 nm, respectively. In these complex biological fluids, nanoparticles can form large aggregates due to changes in pH, ionic strength, and the presence of biomolecules that disturb the delicate balance. Fig. 5 shows the HRTEM images of the Pure and modified NPs. Crystalline fringes in the HRTEM images shows
Fig. 9. FTIR spectra of (a) T2 (b) T3 (c) T4 (d) T5 (e) T6 and (f) T7.
with Rathod P B et al. [38]. He reported the maximum absorbance of titanium dioxide NPs are found to lie in between 220 and 320 nm. Hasan et al. [39] pointed out that the absorbance at 337 nm could be
Fig. 10. Toxicity profiles (concentration vs cell viability) of the KB cells treated with T1, T2, T3 T4, T5, T6 and T7 samples for various concentrations [Cells were treated with different concentrations of the samples for 24 h. At the end of the incubation period, cell viability was determined by MTT assay]. 6
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Fig. 11. Inverted Microscope images of the KB oral cancer cells treated with 10 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
Fig. 12. Inverted Microscope images of the KB oral cancer cells treated with 20 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
C]C stretching vibrations respectively. Fig. 7 shows the broad peak at 633 cm−1 that denotes the O–Ti–O vibration bands [44]. The peaks at 1633 and 3420 cm−1 are the characteristic vibrations of hydroxyl groups. From Fig. 9, it is observed that there is a slight shift at 1635 cm−1 for Clove modified NPs when compared with 1633 cm−1 of Pure NPs. This confirms that there is a chemical reaction between the eugenol in Clove and titanium dioxide nanoparticles. In a similar way, a peak is observed at 1636 cm−1 for Elachi and Cinnamon respectively.
the good crystalline nature of the TiO2 samples. 3.4. FTIR spectra analyses Figs. 7–9 shows the FTIR spectral analyses of pure TiO2, extracts (Clove, Elachi, Cinnamon, Clove - Elachi, Elachi - Cinnamon, Cinnamon - Clove) and bio modified TiO2 NPs [T2, T3, T4, T5, T6 and T7]. From Fig. 8a, the key component of Clove, eugenol shows the characteristic band at 1607 cm−1 due to the C]C stretching vibrations [41]. In the case of Elachi and Cinnamon, the main constituents are 1,8-cineole and cinnamaldehyde. The characteristic peak of cineole [42] and cinnamaldehyde [43] occurs at 1619 cm−1 and 1616 cm−1 which represent
3.5. Anticancer activities of pure and modified samples Fig. 10 shows the anticancer effect of pure, Clove, Elachi, 7
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Fig. 13. Inverted Microscope images of the KB oral cancer cells treated with 30 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
Fig. 14. Inverted Microscope images of the KB oral cancer cells treated with 40 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
50.67%, 20.30%, 4.60%, 6.77%, 14.65%, 13.62% and 21.25% viabilities respectively. Among the modified samples T3 shows excellent anticancer activities when compared with pure and other modified samples. There is a decrease in the band gap of the T3 sample which is responsible for the production of superoxide radicals. This shows that T3 sample releases a greater number of free radicals which is responsible for Reactive Oxygen species (ROS) generation. This ROS is responsible for the destruction of the cancer cells. The component 1,8cineole present in Elachi may increase the anticancer nature in comparison with the pure Titanium dioxide NPs. The anticancer activity increases with the increase in the concentration of the sample. This can be explained through the phenomena that when the concentration increases more number of the NPs penetrate the cells easily and produce more number of superoxide radicals
Cinnamon, Clove - Elachi, Elachi - Cinnamon and Cinnamon - Clove modified TiO2 NPs against KB oral cancer cell line. Five different concentrations are used to analyze the anticancerous nature of the samples. Camptothecin is the standard drug used for the analysis. It is observed that variable cell viability is seen for various samples with same concentration and the bio modified samples enhance the anticancerous nature when modified with the pure sample. From the Figures, it is observed that for 10 μg/mL of T1, T2, T3, T4, T5, T6 and T7 samples showed 98.28%, 81.91%, 44.53%, 31.34%, 70.86%, 55.69% and 55.18% viabilities, for 20 μg/mL showed 77.32%, 65.89%, 23.75%, 18.78%, 49.01%, 42.41% and 43.70% viabilities, for 30 μg/mL showed 69.73%, 45.41%, 12.82%, 15.71%, 32.04%, 25.10% and 35.38% viabilities, for 40 μg/mL showed 60.07%, 35.13%, 5.51%, 11.83%, 21.33%, 20.13% and 28.19% viabilities and for 50 μg/mL showed 8
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Fig. 15. Inverted Microscope images of the KB oral cancer cells treated with 50 μg/mL concentration for (a) Untreated (b) Camptothecin (Control) (c) T1 (d) T2 (e) T3 (f) T4 (g) T5 (h) T6 and (i) T7 samples.
Fig. 16. Zone of Inhibition in mm of (a) T1 [T24] (b) T2 [ T49] (c) T3 [ T50] (d) T4 [ T51] (e) T5 [T52] (f) T6 [T53] and (g) T7 [T 54] samples against Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus & Streptococcus mutans.
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Fig. 17. Zone of Inhibition in mm of (a) T1 [T24] (b) T2 [ T49] (c) T3 [ T50] (d) T4 [ T51] (e) T5 [T52] (f) T6 [T52] and (g) T7 [T 54] samples against Staphylococcus aureus & Streptococcus mutans.
Syzygium aromaticum is the best anticancer agent for lung cancer. He also pointed out that the eugenol; key component of Clove is acting as a good agent for production of oxide radicals. The cinnamaldehyde present in the Cinnamon can be responsible for the damage of the cancer cells. Meena V et al. [24] studied the anticancerous nature of the cinnamaldehyde from Cinnamon bark against Breast cancer cell line (MCF7). She observed that at 200 μg/mL concentration of the sample damaged 32.3% cancer cells. Figs. 11–15 represents the microscopic images of Oral cancer cells with modified plant NPs for different concentrations.
which is responsible for the damage of the cancer cells [45]. Murata S et al. [46] discussed the antitumor effect of 1,8-cineole against colon cancer. He observed that eucalyptol (1,8-cineole) exhibit antitumor activity against HCT116 and RKO (human colon cancer) cell lines when subjected to Western blot analysis and cell viability assay. Moteki H [47] studied the use of cineole on the growth of Molt 4B, HL-60 and KATO III cell lines. He used 5, 7.5 and 10 μm concentration of the cineole and subjected to the cell line for 72 h and then the viability is evaluated using Trypan blue exclusion method. He observed that 1,8cineole exert activity against human leukemia cells but no activity is seen in the human stomach cancer cell line (KATO - III). Ashour H M [48] studied the anti tumor properties of essential oils of Eucalyptus species on MCF7 (Human breast adenocarcinoma) and HEPG2 (Human hepatocellular carcinoma) cell lines. He observed that E.torquata leaves exhibit cytotoxic activity against MCF7 cell lines. The presence of eugenol in the Clove may be responsible for the anticancer nature of the Clove modified TiO2. Dwivedi V et al. [49] studied the anticancer nature of the water, ethyl alcohol and oil extracts of Clove. He showed that maximum death occurs in TE-13, esophageal cancer cell line due to apoptosis. He pointed out that the cytotoxic activities of the cells are due to the presence of eugenol in the Clove extract. This eugenol is responsible for all pharmacological activities and also kills maximum cancer cells. Banerjee S [50] observed that the
3.6. Antibacterial activities of pure and modified samples Fig. 16 and Fig. 17 showed the zone of inhibition for T1, T2, T3, T4, T5, T6 and T7 samples against Gram-positive bacteria such as Staphylococcus aureus, Streptococcus mutans and Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. It is observed that zone of inhibition is maximum for Clove modified [T2] TiO2 NPs against all bacterial Strains. No activity is seen for T4 and T7 samples for any bacterial strains. Maximum zone of Inhibition for T1, T2 and T6 is observed for Gram positive bacteria such as Streptococcus mutans. Similarly, for T3 and T5 samples maximum zone is seen against Pseudomonas aeruginosa. It is seen that eugenol 10
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Fig. 18. Graph for antibacterial activities of (a) T1 (b) T2 (c) T3 (d) T4 (e) T5 (f) T6 and (g) T7 samples against Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus & Streptococcus mutans.
reported about the elements present and their pharmacological activities of the Syzygium aromaticum essential oil extracted from the dried flower buds of clove. He observed eugenol to be one of the major chemical constituents. The oil is subjected to Agar disk diffusion method for antibacterial activity and Gentamicin is used as a standard antibiotic and zone of inhibition is measured by using bacterial strains. The maximum zone is observed against S. epidermidis and this maximum activity may be due to the presence of eugenol in the sample. Kumar Y [19] compared the antibacterial nature of the Clove and Garlic extracts. He also pointed that Clove showed 23, 15 and 10 mm inhibition zone against Salmonella typhi at 2000, 1500 and 1000 ppm at concentration. The T1 NPs produces more amount of reactive oxygen species and they easily enter inside the antibacterial activities when compared with other pure and modified sample. The biomolecules present in the Clove modified TiO2 enhances the antibacterial activity of the samples. Fig. 19 represents graphical representation of biological applications of the samples.
present in the Clove may be responsible for the biological activities, Fig. 18 represents the Graphical representation of antibacterial activities of pure and spices modified samples against Gram positive and Gram negative bacterial organisms. Burt S [51] in his review article pointed out that eugenol, present in the Clove is responsible for the breaking up of the cells which in turn damages the cell wall of the Bacillus cereus. Sharma A et al. [52] studied the antimicrobial nature of the aqueous Clove and Ginger extracts against Streptococcus mutans. He observed that 5% of Clove extract shows no activity whereas 50% of extract showed excellent activity against S. mutans. He studied the extracts of Clove and Ginger for antimicrobial nature and found that Clove extracts showed more antibacterial nature when compared with that of the Ginger extracts. Xu J G et al. [53] did his research with essential oil of Clove for the determination of its antibacterial nature. He reported that eugenol forms the major constituent of the Clove (72.3%) and Clove is found to destroy the bacterial cell wall and membrane. At the same time, it stops the normal procedure for the activation of DNA and proteins that are important for the growth of bacteria. Liu Q et al. [15] studied about the antibacterial and antifungal activity of the various spices such as Syzygium aromaticum, Origanum vulgare, Thymus vulgaris, Cinnamomum verum and Cuminum cyminum against food spoilage pathogens like Bacillus subtilis and Pseudomonas fluorescens and Staphylococcus aureus. He concluded that spices can become new and safer antimicrobial agents. Chaeib K et al. [18]
4. Conclusion The Syzygium aromaticum, Elettaria cardamomum and Cinnamomum verum modified NPs were synthesized using hydrothermal method and characterized using XRD, UV, FTIR and TEM analysis. The research experiments analysed the biological activities of the synthesized samples which confirmed that the modifiers have enhanced the biological 11
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Fig. 19. Graphical representation of Biological applications.
activities of the TiO2 NPs. The inhibition bacterial zones are found to be maximum for Syzygium aromaticum modified TiO2 samples in comparison with the pure samples against Gram positive and Gram negative bacterial strains. The Elachi modified TiO2 NPs showed minimum cell viability among the cancer cells at 50 μg/mL concentration of the sample. Hence from the research findings, it is concluded that modified samples are found to be an excellent anticancer and antibacterial agent when compared with the pure samples.
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