Physical properties, biological applications and biocompatibility studies on biosynthesized single phase cobalt oxide (Co3O4) nanoparticles via Sageretia thea (Osbeck.)

Ali Talha Khalil; Muhammad Ovais; Ikram Ullah; Muhammad Ali; Zabta Khan Shinwari; Malik Maaza

doi:10.1016/j.arabjc.2017.07.004

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Original article

13 (

); 606-619

doi:

10.1016/j.arabjc.2017.07.004

Physical properties, biological applications and biocompatibility studies on biosynthesized single phase cobalt oxide (Co₃O₄) nanoparticles via Sageretia thea (Osbeck.)

Ali Talha Khalil^{^a,^c,^d,⁎}, Muhammad Ovais^{^a}, Ikram Ullah^{^a}, Muhammad Ali^{^a}, Zabta Khan Shinwari^{^a,^b}, Malik Maaza^{^c,^d}

a Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan

b Pakistan Academy of Sciences, Pakistan

c UNESCO-UNISA Africa Chair in Nanosciences and Nanotechnology, College of Graduate Studies, University of South Africa, Pretoria, South Africa

d Nanosciences African Network (NANOAFNET), Materials Research Department, iThemba Labs, Westren Cape, South Africa

⁎Corresponding author at: Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan. talhakhalil.qau@gmail.com (Ali Talha Khalil)

Received: 2017-5-17, Accepted: 2017-7-10, Published: 2017-01-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

Cobalt oxide nanoparticles were successfully biosynthesized by complete green process using aqueous leaf extracts of Sageretia thea as chelating agent. Diverse techniques were applied for characterization. Antibacterial (with and without UV illumination), antileishmanial, antioxidant and enzyme inhibition applications were assessed, while freshly isolated macrophages and red blood cells were used for biocompatibility studies. Good antibacterial nature and enhancement of bactericidal nature upon UV modulation is reported. Staphylococcus aureus and Escherichia coli are indicated as most susceptible bacterial strains. Significant cytotoxic potential is revealed with IC₅₀ calculated as 12.82 µg/ml and 3.16 µg/ml against the axenic leishmanial promastigote and amastigote cultures respectively. Biogenic cobalt oxide nanoparticles indicated DPPH free radical scavenging potential, while moderate antioxidant capacity and reducing power was demonstrated. Bioinspired cobalt oxide also demonstrated alpha amylase and protein kinase inhibition at higher concentrations. Biogenic cobalt oxide was found as more cytotoxic to macrophages (IC₅₀ = 58.55 µg/ml) then to RBC’s (IC₅₀>200 µg/ml). Our results indicate green synthesis as an alternative, effective and eco-friendly method for the biosynthesis of cobalt oxide nanoparticles with numerous biological applications.

Keywords

Biosynthesis

Cobalt oxide

Nanoparticles

Antimicrobial

Antileishmanial

Antioxidant

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1 Introduction

Bioinspired and multifunctional metal and metallic oxide nanoparticles are considered as a bright area of research because of their exciting physio-chemical and optoelectronics properties (Thema et al., 2016, 2015b,a; Thovhogi et al., 2016, 2015; Ismail et al., 2016; Diallo et al., 2016; Matinise et al., 2017; Sone et al., 2019; Diallo et al., 2015b,a; Sone et al., 2015; Ezhilarasi et al., 2016 ). Metal oxide nanoparticles has been frequently synthesized and tested in a wide range of applications. Rapid developments on the nano-biotechnological interface has resulted in a wide array of biomedical applications including drug delivery and vaccine administration (Khan et al., 2015a,b). Nanoparticulate matter is different from their microscale counter parts in their magneto-optical, electro-optical, mechanical, chemical and surface area to volume ratio which signify them as an effective tool for biomedical applications. Cobalt oxide possess interesting properties and therefore has attracted numerous researchers for studying their possible biomedical applications (Wang et al., 2005). Beside their physiological role as a cofactor of vitamin B12, cobalt can be used in a wide range of applications.

Cobalt oxide is a multifunctional, antiferromagnetic p-type semiconductor (with a direct optical bandgap of 1.48 and 2.19 eV) (Raman et al., 2016) has been used in electrochromic sensors, energy storage, heterogeneous catalysis, pigments, dyes, and in lithium ion rechargeable batteries as an anode material (Askarinejad et al., 2010; Li et al., 2005; Shinde et al., 2006; Kaviyarasu et al., 2013; Diallo et al., 2015a ). Cobalt based nanostructures have been successfully used in methanol, glucose, nitrites and amino acids (Yang et al., 2006; Shen et al., 2008; Adekunle et al., 2010; Song et al., 2011 ). Because of their interesting physical properties, cobalt oxide also have spintronic applications (Diallo et al., 2015a).

Many physical and chemical techniques has been applied for the synthesis of cobalt oxide nanoparticles. Hydrothermal reaction, thermal decomposition, solution combustion, microwave assisted, micro-emulsion method, chemical spray pyrolysis and vapor deposition method, sono-chemical, co-precipitation and other mechano-chemical processes has been applied for cobalt oxide nanoparticles synthesis (Salavati-Niasari et al., 2009; Dong et al., 2014; Thota et al., 2009; Makhlouf et al., 2013; Zhang et al., 2012; Dai et al., 2013; Farhadi et al., 2013 ). Hitherto, being effective, the aforementioned synthesis methods are accompanied by certain disadvantages like being costly, time and energy consuming and being environment unfriendly. To overcome the problem of toxic wastes and energy imbalance, greener and ecofriendly methods have been proposed (Diallo et al., 2015a). Biological resources such as plants and microorganisms can be used in a rapid, effective, simple and economical way to produce the desired metal or metallic oxide nanoparticles (Ovais et al., 2016, 2017). Plant mediated biosynthesis of cobalt oxide nanoparticles has been successfully demonstrated (Diallo et al., 2015a).

With the aim of synthesizing cobalt oxide nanoparticles, a complete green approach was adopted using aqueous leaf extracts of medicinal plant Sageretia thea (Osbeck.), as an effective stabilizing and chelating agent. The fact that there was neither the use of organic/inorganic solvents and nor the use of any surfactants making the process as an ecofriendly and green. The interface of medicinal plants and biosynthesis of nanoparticles provides an exciting opportunities for wide range of biomedical applications. Hence, the as synthesized cobalt oxide nanoparticles were further investigated for their possible biological applications and biocompatibility with human blood cells.

2 Material and methods

2.1

2.1 Plant material processing

Sageretia thea was collected from Islamabad, Pakistan and taxonomically verified in Department of Plant Sciences, Quaid-i-Azam University (QAU), Islamabad. The herbarium specimen with voucher number MOSEL-343 was deposited at Molecular Systematics and Applied Ethnobotany Lab (MoSAEL), Department of Biotechnology, QAU. Fresh leaves were excised and grounded to fine powder in a Willy mill. Aqueous extraction was performed on the plant powder, while remaining was stored for further use. In Fig. 1 the overall outline of the study has been summarized.

2.2

2.2 Biosynthesis procedure

Already established procedure for the biosynthesis of metal oxide nanoparticles was used (Thema et al., 2015a). Leaf extracts were obtained by adding 200 ml of deionized water to 30 g of powdered plant material, followed by heating at of ∼80 °C on a magnetic stirring hot plate (Snijders) for 1 h. The resultant extract solution was filtered thrice with Whattman filter paper to remove solid residual waste. To 100 ml of filtered solution 6.0 g of the precursor salt i.e. cobalt acetate was added. pH change was observed from 5.7 to 4.5 after precursor addition. The solution was heated for 2 h at ∼60 °C with gentle stirring. The solution was allowed to cool to room temperature and centrifuged (10,000 rpm/10 min) to collect the pellet that was subsequently washed 3 times with deionized water. Obtained pellet was dried at 100 °C for 2 h, followed by annealing at 500 °C in open air for obtaining highly crystalline pure phase cobalt oxide nanoparticles. A brief biosynthesis mechanism has been indicated in Fig. 2.

Plausible mechanism of biosynthesis cobalt oxide nanoparticles using Sageretia thea aqueous extracts.

2.3

2.3 Characterization

X-ray diffractometer (model Bruker AXS D8 Advance) equipped with irradiation line Kα of copper (λ = 1.5406 Å) was used to record the XRD spectrum and their corresponding size was calculated using Scherer equation {〈Ø_size〉 = K λ/Δθ_1/2 cos θ}. Vibrational properties were studied using ATR-FTIR (400–4000 cm⁻¹) and raman spectroscopy over the range from 0 to 1000 cm⁻¹. Raman spectrum was recorded using a laser line of 473 nm with average excitation power of 2.48 mW. Morphology and shape was studied via HR-SEM and HR-TEM, while particle distribution was investigated after digitizing the various HR-TEM images using image J software the morphological investigation was carried out through HR-SEM and HR-TEM. In addition, Selected Area Electron Diffraction (SAED) and Energy Dispersive X-ray spectroscopy (EDS) were also carried out.

2.4

2.4 Antibacterial potential

Previously described disc diffusion method (Fatima et al., 2015) was used for investigating the antibacterial nature of bioinspired cobalt oxide nanoparticles, while broth dilution assay was used to determine their MIC. Bacterial strains already available at the department of biotechnology were refreshed on nutrient agar (Oxoid-CM0003) before the assay. Bacterial cultures were inoculated to nutrient broth, and grown in shaker incubator to the optical density of 0.5 at 600 nm which corresponds to 1 × 10⁸ CFU/ml. 100 µl of broth cultures were dispensed in culture plates and uniform microbial lawns were prepared using sterilized cotton swabs. Filter paper discs (6 mm) loaded with 10 µl of the test sample over the concentration range from 1000 µg/ml to 31.25 µg/ml were used to determine the antibacterial activity. Pure gentamycin disc (10 µg) were used as positive control. Zones of inhibition were measured using vernier caliper. Antibacterial potential was also investigated after exposing cobalt oxide nanoparticles to UV for 20 min. For UV-illumination germicidal 6 W UV Lamp 6GT5 (Sankyo denki- Japan) was used.

2.5

2.5 Brine shrimp cytotoxicity

The cytotoxicity of biogenic cobalt oxide nanoparticles was assessed using Artemia salina larvae in a 96 well plate as described previously (Ali et al., 2017; Khan et al., 2015a,b ). After incubation of the various concentrations of test samples with brine shrimps for 24 h, the number of dead shrimps were counted in each well and percent mortality were calculated. IC₅₀ values were calculated using table curve software.

2.6

2.6 Antileishmanial activity (Promastigotes and Amastigotes)

MTT cytotoxic activity (Ali et al., 2017) was carried out against the axenic promastigote and amastigote cultures of Leishmania tropica KWH23 for determining the antileishmanial potential. M199 media supplemented with 10% fetal bovine serum (FBS) was used for culturing the parasite while their density was maintained at 1 × 10⁶ cells/ml for MTT assay. 96 well microplate was used to test bioinspired cobalt oxide nanoparticles over concentration range from 200 µg/ml to 1 µg/ml. Amphotericin B and DMSO were used as positive and negative control. Median lethal concentration (IC₅₀) was calculated using table curve software. The seeded 96 well microplate was incubated at 24 °C for 72 h. Readings were taken at 540 nm, while percent inhibition was calculated using the following formula; $% Inhibition = [1 - {Sample absorbance} \div {Absorbance of control}] \times 100$

2.7

2.7 Biocompatibility assessment

2.7.1

2.7.1 Biocompatibility with macrophages

MTT cytotoxic assay (Ali et al., 2017) was performed on the freshly isolated human macrophages to study the compatibility of biosynthesized cobalt oxide nanoparticles. Macrophages were isolated from peripheral human blood through ficoll-gastrografin (density = 1.070 g/ml) density gradient method as described previously (de Almeida et al., 2000). Briefly, 5 ml of gastrografin was added to the 95 ml of deionized water, while 5.7 g of ficoll was slowly added. Blood was diluted with Hank’s buffer salt solution (HBSS) followed by gentle layering on ficoll-gastrografin. The solution was further centrifuged for 30 min at 400g followed by purification with percoll gradient (density 1.064g/ml) adjusted with sterilized deionized water. Macrophages were suspended in RPMI medium supplemented with fetal bovine serum (10%), Hepes (25 mM), and antibiotics (Streptomycin: 0.1 mg/ml; Penicillin:100 U/ml). Cells were kept in humified incubator to grow to a density of 1 × 10⁵ cells/well. Percentage inhibition was calculated using formula; $% inhibition = [1 - {sample absorption} \div {absorbtion of control} \times 100$

2.7.2

2.7.2 Hemolytic assay

To further assess the biocompatibility against human RBC’s, hemolytic assay was carried out as described previously (Malagoli, 2007). Fresh blood was isolated and dispensed in EDTA tube. RBC’s were isolated by centrifugation of blood at 14,000 rpm for 5 min. Erythrocytes suspension was prepared by dispensing 200 µl of the isolated pellet to 9.8 ml phosphate buffer saline (pH:7.2). Hemolytic activity was determined by adding 100 µl of test concentration of cobalt oxide nanoparticles with 100 µl of erythrocytes suspension, followed by incubation for 1 h at 35 °C. This was followed by further centrifugation at 10,000 rpm for 10 min and supernatant was dispensed in the 96 well microplate reader to monitor the percent hemoglobin release at 540 nm. Triton X-100 and DMSO were used as positive and negative controls respectively. Percentage hemolysis induced by the nanoparticles was calculated through the formula; $Haemolysis = [{Sample (abs) - Neg . control (abs)} \div {Pos . control - Neg . control (abs)}] \times 100$

2.8

2.8 Antioxidant activities

2.8.1

2.8.1 DPPH radical scavenging

Spectrophotometric method (Ali et al., 2017) was employed to investigate the quenching ability of bioinspired cobalt oxide nanoparticles. DPPH is a stable free radical widely used to test the radical scavenging ability of samples. DPPH free radical scavenging was investigated in the concentration range of 200 µg/ml to 1 µg/ml. Ascorbic acid and DMSO were used as positive and negative controls. The reaction mixture (final volume 200 µl) comprised of 20 µl of test sample and 180 µl of reagent. After incubation for 20 min in dark, absorbance was measured at 517 nm and percent radical scavenging was calculated as; $% DPPH radical scavenging = [1 - {Sample absorbance} \div {Absorbance of control}] \times 100$

2.8.2

2.8.2 Reducing power

Potassium ferricyanide [K₃Fe (CN)₆] based method (Javed et al., 2016) was used to determine total reducing power of biogenic cobalt oxide nanoparticles. Ascorbic acid and DMSO were used as positive and negative controls respectively. Absorbance was measured at 630 nm and the results were expressed as ascorbic equivalents per mg of sample.

2.8.3

2.8.3 Total antioxidant capacity

Phosphomolybdenum based method (Jafri et al., 2017) was used to determine total antioxidant capacity. Readings were taken at 695 nm and results were expressed as number of ascorbic acid equivalents in µg per mg of the sample i.e. µg AAE/mg.

2.9

2.9 Enzyme inhibition assays

2.9.1

2.9.1 Protein kinase inhibition

PK inhibition was investigated using Streptomyces 85E strain as described previously (Fatima et al., 2015). ISP4 minimal media was used to produce uninform lawns of Streptomyces 85E strain with pre-adjusted optical density of 0.5 at 600 nm. Surfactin and DMSO were used as positive and negative control. 6 mm filter disc loaded with 10 µl of test sample were gently placed on the uniform lawns. Seeded plates were incubated at 30 °C for 72 h. Zones of inhibition were measured using vernier caliper.

2.9.2

2.9.2 Alpha amylase inhibition

Alpha amylase inhibition assay was performed in 96 well plat as described previously (Ali et al., 2017). The reaction mix (15 µl PBS/25 µl α-amylase enzyme) was added with test samples (10 µl) and starch solution (40 µl) step wise and incubated at 50 °C for 30 min. After incubation, 20 µl of 1 M HCl and 90 µl of iodine solution were added to the reaction mix. Blank solution comprised of PBS deionized water and starch, while negative and positive control comprised of DMSO and acarbose respectively. Readings were taken at 540 nm and the enzyme inhibition was calculated by following equation; $Enzyme inhibition = [{{OD}_{S} - {OD}_{N}} \div {{OD}_{B} - {OD}_{N}}] \times 100$ where “OD_S”_, “OD_N” and “OD_B” corresponds to the optical densities of sample, negative control and blank respectively.

3 Results and discussion

Although, the biosynthesis of cobalt oxide nanoparticles have rarely been done in previous reports, but their antimicrobial, antileishmanial, antioxidant and enzyme inhibition has never been investigated. Hence it is worthy to mention that this report will be the first of such kind that has successfully produced cobalt oxide nanoparticles via green route but also presented a broad overview on the possible biomedical applications of green cobalt oxide nanoparticles.

3.1

3.1 Biosynthesis of cobalt oxide nanoparticles

Physical or chemical means for synthesis of nanoparticles possess certain disadvantages such as being laborious, hazardous and time consuming. Even it was indicated that certain toxic chemicals used in chemical synthesis can retain on the nanoparticle surface which limits their biomedical applications. On the contrary, green synthesis using medicinal plants is deprived of such disadvantages therefore has been preferred in the present report. A complete green route for the biosynthesis of cobalt oxide nanoparticles was successfully optimized using aqueous extracts of leaf of Sageretia thea. Medicinal uses of Sageretia thea (Bird Plum/English) are well documented. It is used in the treatment of hepatitis, jaundice, circulatory and cardio-vascular diseases. Leaves are used in making tea in parts of Korea and China (Hyun et al., 2015; Khan et al., 2014; Murad et al., 2011 ). Bioactive compounds like Taraxerol, Quercetin, Syringic acid, Myricetrin, Kaempferol, Daucosterol have previously been reported from S. thea (Shen et al., 2009; Chung et al., 2004; Xu et al., 1994 ). Such phytochemical components has an intended role in stabilizing of nanoparticles (Park et al., 2011). A schematic representation of biosynthesis procedure is indicated in Fig. 2.

3.2

3.2 Physical characterization

Crystalline, vibrational and morphological properties of biosynthesized cobalt oxide were studied. XRD spectrum confirmed the formation of crystalline and single phase of cobalt oxide nanoparticles. The observed Bragg peaks were the crystallographic reflections of the single phase, face centered cubic cobalt oxide belonging to the space group Fd3m. Brag peaks were found consistent with which are consistent with the JCPDS pattern no. 00-042-1467. The average lattice parameters were investigated as 〈a_exp〉 = 0.807 nm and 〈a_bulk〉 = 0.808 nm. Average size was calculated as 20.03 nm using Debye Scherer approximation. No other peaks of related cobalt based compounds were indicated which affirms the purity of the single phase cobalt oxide. Results from the XRD analysis are summarized in Fig. 3(A/B). Morphology of the bioinspired cobalt oxide nanoparticles was studied using HR-TEM and HR-SEM. The inset of Figs. 4 and 5 suggest various HR-TEM and HR-SEM images. Cubic morphology of the unit cell can be deduced from HR-TEM images (Fig. 4B) which are in line with the data obtained from XRD analysis. HR-SEM images indicate a certain degree of agglomeration. Particle distribution (Fig. 5D) was calculated after the digitization of various HR-TEM images. The selected area electron diffraction studies further confirms the crystalline nature of the biosynthesized cobalt oxide nanoparticles as indicated in Fig. 4F. The elemental phase of biosynthesized nanoparticles were further assessed using Energy Dispersive X-ray Spectroscopy (EDS) as indicated in Fig. 6 (A). EDS analysis indicate the presence of cobalt and oxygen, while the presence of carbon is attributed to the grid support.

XRD analysis of biosynthesized cobalt oxide nanoparticles; (A): XRD spectra; (B): Average size calculation using Scherer size approximation formula.

Various HR-TEM images of biosynthesized cobalt oxide nanoparticles; (A/B/C/D): size distribution and shape; (E): HR-TEM image; (F): Particle size distribution.

Scanning electron microscopy of biosynthesized cobalt oxide nanoparticles; (A/B/C): Various HR SEM images; (D): Particle size distribution.

Spectral studies of biogenic cobalt oxide; (A) EDS spectra of biosynthesized cobalt oxide nanoparticles; (B) Raman spectra of biosynthesized cobalt oxide nanoparticles; (C) ATR-FTIR spectra of biosynthesized cobalt oxide nanoparticles.

The vibrational properties were studied through Raman spectroscopy and ATR-FTIR. For Fd3m symmetry, the group theory predicts the following active modes; A_1g (R) + E_g (R) + F_1g (IN) + 3F_2g (R) + 2A_2U (IN) + 2E_U (IN) + 4F_1U (IR) + 2F_2U (IN) whereas (R), (IR) and (IN) depicts Raman active vibrations, infrared active vibrations and inactive modes. Fig. 6(B) indicate the room temperature Raman spectra of the biogenic cobalt oxide nanoparticles. One can single out 6 different Raman active modes centered at ∼195, ∼369, ∼550, ∼698, ∼750 and ∼838 cm⁻¹. Peaks centered at ∼195 cm⁻¹, 550 cm⁻¹ and 698 cm⁻¹ are in agreement with some of the earlier studies (Diallo et al., 2015a; Jakubek et al., 2015), and therefore confirm cobalt oxide nanostructures. Positioning of Raman peaks, however can change with synthesis methods and distribution of vacancies within the unit cell (De Faria et al., 1997). To further validate the cobalt oxide nature of the biosynthesized nanoparticles, ATR-FTIR analysis were carried out in the spectral range of 400–4000 cm⁻¹, as indicated in Fig. 6(C). Major IR abortions were singled out by plotting IR optical transmission versus log of wavenumbers. Two sharp IR bands were observed centered at 576 cm⁻¹ and 674 cm⁻¹. These absorption modes are attributed to the fingerprint stretching vibrations of Co—O bond in Co₃O₄ (Ren et al., 2009). The IR absorption observed at 576 cm⁻¹ is the vibration from O—Co, with cobalt depicting the Co⁺³ in octahedral site. Absorption band at 674 cm⁻¹ is attributed to the Co²⁺ Co³⁺O₃ (Co²⁺ is tetrahedral site) vibrations in the spinal lattice (Ai and Jiang, 2009; Salavati-Niasari et al., 2009).

3.3

3.3 Antibacterial activities

As synthesized cobalt oxide nanoparticles were studied for their antibacterial potential against 3 g negative (Pseudomonas aeruginosa, Klebsiella pneumonia, and Escherichia coli) and 3 g positive bacterial strains (Staphylococcus epidermis, Staphylococcus aureus and Bacillus subtilis). It was noted that the antibacterial potential increased with increase in the concentration of the nanoparticles. Similarly, upon UV modulation the antibacterial activities showed enhancement. Staphylococcus aureus and Escherichia coli were found as the most susceptible strains with MIC and MIC^uv as 31.25 and 31.25 µg/ml respectively. Pseudomonas aeruginosa was found to be the least susceptible strain with MIC and MIC^uv as 250 and 62.5 µg/ml respectively. The antibacterial response was concluded to be dose dependent, while none of the tested samples were found more effective then gentamycin antibiotic disc (10 µg). The control antibiotic indicated significantly higher zones of inhibition as compared to test bioinspired cobalt oxide samples. Previous study (Khan et al., 2015a,b) also indicated cobalt oxide nanoparticles synthesized via chemical route as less effective than the standard drugs which is in agreement to our findings, however the same study reports MIC > 10,000 µg/ml for cobalt oxide nanoparticles which is in disagreement to our findings. Here, it can be suggested that the disagreement in the results are because of the difference in synthesis methods. While using medicinal plants as a stabilizing and capping agents, it can be inferred that some of the bioactive phenolic components are used in capping of the nanoparticles (Gatselou et al., 2016; Durán et al., 2011) and those components remains capped to the nanoparticles. Those phenolic compounds can be the result in the enhancement of the antibacterial potential of biogenic cobalt oxide relative to chemically produced counterparts. The antibacterial activities across different test concentrations are summarized in Fig. 7(A–C) while there minimum inhibitory concentrations are indicated in Table 1.

Antibacterial activities; (A): without UV-illumination; (B): with UV- illumination.

Table 1 Minimum inhibitory concentration of biogenic cobalt oxide nanoparticles against various bacterial strains.

Without UV illumination		With UV illumination
Gram positive		Gram positive
Bacterial strain	MIC (µg/ml)	Bacterial strain	MIC (µg/ml)
Staphylococcus aureus	31.25	Staphylococcus aureus	31.25
Staphylococcus epidermis	125	Staphylococcus epidermis	31.25
Bacillus subtilis	125	Bacillus subtilis	31.25

Gram negative		Gram negative
Klebsiella pneumonia	62.5	Klebsiella pneumonia	31.25
Pseudomonas aeruginosa	250	Pseudomonas aeruginosa	62.5
Escherichia coli	31.25	Escherichia coli	31.25

3.4

3.4 Cytotoxic activities

3.4.1

3.4.1 Brine shrimp cytotoxicity

Preliminary cytotoxicity of biogenic cobalt oxide nanoparticles was confirmed through the brine shrimp cytotoxicity assay. Brine shrimps are widely used as a reference organism for screening the cytotoxic potential of chemical entities. Fig. 8(A) indicate the cytotoxicity of biosynthesized cobalt oxide nanoparticles towards brine shrimps. Cytotoxicity of the cobalt oxide nanoparticles was confirmed by their dose dependent response while the median lethal concentration was calculated as 19.18 µg/ml.

Cytotoxicity of cobalt oxide nanoparticles; (A): Cytotoxicity against brine shrimps and leishmania; (B): Biocompatibility with RBC’s and macrophages; (C) Median lethal concentrations.

3.4.2

3.4.2 Antileishmanial activities

Leishamniasis is deadly disease which comes under the category of neglected tropical disease, is endemic to ∼100 countries with about ∼350 million people living under the direct threat. To date, there are not as such effective antileishmanial drugs while the current treatment medications are accompanied by disadvantages like side effects, cost, toxicity and long therapy duration (Abamor, 2017). Antimonials were used as a gold standard therapy for leishmania, which however has lost their effectiveness because of drug resistance (Hadighi et al., 2006). Therefore, the treatment for leishmania consequently requires an alternative approach. Metal oxide nanoparticles recently demonstrated their abilities in considerably reducing the leishmania population in invitro studies (Ali et al., 2017; Nadhman et al., 2016). In the present study, the antileishmanial nature of the biogenic nanoparticulate cobalt oxide has been assessed using MTT cytotoxic assay for the first time. Both of the axenic promastigote and amastigote cultures were found highly susceptible to the tested concentrations. Amastigote leishmania were found more susceptible (IC₅₀ = 3.16 µg/ml) relative to promastigote (IC₅₀ = 12.82 µg/ml). The life cycle of leishmania is simple digenetic i.e. Leishmania exist in 2 forms (promastigote and amastigote). The amastigotes are present inside the body as circular and non-flagellated forms while the motile promastigotes are found outside the human body (Zilberstein et al., 1991). It was found that the antileishmanial response was dose dependent. Our results further indicate the possible applications of biogenic cobalt oxide nanoparticles in nanomedicine for the treatment of leishmania at any stage of its life cycle. Results are indicated in Fig. 8(A).

3.5

3.5 Biocompatibility assessment

In response to the potential inhibitory effects of biogenic cobalt oxide on microorganisms, it was considered imperative to investigate their biocompatibility with other normal human cells. Henceforth the biocompatibility was assessed under invitro conditions, using freshly isolated red blood cells and macrophages. Results about the percent hemolysis are indicated in Fig. 8(B). MTT cytotoxicity was carried out against freshly isolated macrophages and the results indicated their toxicity towards macrophages. Median lethal concentration were investigated as >200 µg/ml and 58.55 µg/ml for RBC’s and macrophages respectively. Therefore, it can be concluded that biogenic cobalt oxide nanoparticles can be used in therapies at low concentrations. However, these results are preliminary, and further research is recommended on the compatibility of bioinspired cobalt oxide nanoparticles with normal human cells. The results of the cytotoxic activities are summarised in Table 2.

Table 2 IC₅₀ values of biogenic cobalt oxide nanoparticles.

Assay type	IC₅₀ (µg/ml)
Antileishmanial promastigotes	12.82
Antileishmanial amastigotes	3.16
Brine shrimp cytotoxicity	19.18
Human macrophages	58.55
Human RBC’s	>200
Alpha amylase inhibition	>200

3.6

3.6 Mechanism of cytotoxicity

Taken together, the bioinspired cobalt oxide nanoparticles manifested toxicity. Researchers have proposed different mechanisms for the cobalt oxide mediated cytotoxicity. Recently described Trojan horse is considered one of the mechanism by which cytotoxicity is manifested by cobalt oxide nanoparticles. The endocytosis mechanism for nanoparticles entry to the cells is well established (Contreras et al., 2010; Bregar et al., 2013) however recent research has indicated the uptake of cobalt oxide nanoparticles via non-endocytotic pathway (Li and Malmstadt, 2013; Lin and Alexander-Katz, 2013). After entering the cells, the cobalt nanoparticles can leach Co⁺⁺ ions that can lead to impairment in the nearby cellular organelle by a trojan horse like mechanism (Ortega et al., 2014). Similarly, as with case with other metal nanoparticles, the ROS generation in one of the primary cause of cellular disruption by oxidative stress. Active nanoparticle surface of cobalt oxide can readily generate ROS species by interactive with oxygen (Limor et al., 2011). Double strand DNA breaks are also reported for cobalt oxide nanoparticles (Uboldi et al., 2016). A detailed schematic on the cytotoxicity mechanisms and pathways is presented in Fig. 9.

Schematic of the potential cytotoxic mechanisms of biogenically synthesized cobalt oxide nanoparticles; (1) indicate the ROS generation by interaction with oxygen; (2) indicate the Trojan horse like process in which Co++ ions are generated in an acidic medium which can interfere with the cellular organelle; (3) Membrane damage; (4/5) Interaction of ROS, Co++ ions and cobalt oxide nanoparticles with cellular proteins and enzymes; (6) indicate interaction of nanoparticles with nuclear material leading to DNA fragments; (7) Trojan horse like process in cellular organelle with acidic media; (8) Mitochondrial disruption by various ROS and cobalt oxide nanoparticles. Note: These schematic is drawn by studying various mechanism of nanoparticles mediated cytotoxicity.

3.7

3.7 Antioxidant assays

Antioxidant potential of the bioinspired cobalt oxide was studied and results are indicated in 10. Free radical scavenging, total antioxidant capacity and total reducing power potential were studied. Highest DPPH radical scavenging (57%) was observed at 200 µg/ml while the scavenging ability decreased at lower concentrations. Similar trends were also observed with total antioxidant activity and total reducing power. Total reducing power and total antioxidant capacity were highest at 200 µg/ml, i.e. 19.8 µg AAE/mg and 23.6 µg AAE/mg of bioinspired cobalt oxide nanoparticles respectively. Overall, good radical scavenging potential, moderate total antioxidant capacity and total reducing power potential is reported for biogenic cobalt oxide nanoparticles.

Antioxidant assays of biosynthesized cobalt oxide nanoparticles.

3.8

3.8 Enzyme inhibition assays

Fig. 11(A) indicate the alpha amylase enzyme inhibition potential of the as synthesized cobalt oxide nanoparticles. Alpha amylase enzyme catalyze the breakdown of carbohydrates into glucose and therefore has been associated with the postprandial glucose excursion in a patient suffering from diabetes. Hence, alpha amylase enzyme inhibitors has been considered in diabetes research. Moderate inhibition is reported (34%) at the highest concentration (200 µg/ml) while no inhibition is reported at the lowest concentration of (1 µg/ml).

Enzyme inhibition assays; (A): alpha amylase enzyme inhibition; (B): Protein kinase enzyme inhibition assay.

Fig. 11 (B) indicate the protein kinase (PK) enzyme inhibition potential of the biosynthesized cobalt oxide nanoparticles. PK are considered an important area for anticancer research. PK enzymes phosphorylates the serine-threonine and tyrosine amino acid which functions in cellular differentiation, proliferation and apoptosis. Deregulated phosphorylations by PK enzymes at the aforementioned amino acids residues can lead to tumor growth. Such entities capable of PK inhibition are looked for in cancer therapies. PK phosphorylations also play a crucial role in hyphae formation in Streptomyces and therefore this microorganism has been readily used in identification of PK inhibitors. Streptomyces 85E strain was used to screen PK inhibition potential of the as synthesized cobalt oxide nanoparticles. Our results indicate the formation of bald zones up to 50 µg/ml while no zones are reported at concentrations lower than 50 µg/ml. Inhibitory zones were 9.4 mm, 6.1 mm and 4.6 mm at 200, 100 and 50 µg/ml respectively. These results indicate that bioinspired cobalt oxide nanoparticles could be further explored in anticancer therapies via further studies on its protein kinase inhibition mechanism.

4 Conclusion

Although cobalt oxide has a wide range of applications like their use in supercapacitors, pseudocapacitors, sensors, MRI and in biomedicine, however the biological synthesis of nanoscale cobalt oxide has rarely been achieved. A complete green route has been presented for nanoscaled cobalt oxide nanoparticles. Biogenic cobalt oxide nanoparticles indicated a varying degree of antibacterial potential against different pathogenic bacterial strains. Significant antileishmanial potential is reported. In addition, bioinspired cobalt oxide nanoparticles were found as more biocompatible to RBC’s then to macrophages. Significant dpph radical scavenging is reported. Moderate antioxidant, reducing power and enzyme inhibition activities are reported. By summarizing our results, it can be suggested that cobalt oxide can be used for treatment of various diseases especially leishamniasis. However, we recommend further studies on the biocompatibility and toxicity of the cobalt oxide nanoparticles towards normal human cells. Moreover, studies should be undertaken on the mechanistic aspects of the biosynthesis of nanoparticles.

Acknowledgments

Authors are indebted to the fellows of Molecular Systematics and Applied Ethnobotany Lab (MoSAEL), Department of Biotechnology, QAU, Islamabad and fellows of Material Research Department (MRD)- iThemba labs Cape town, South Africa. All authors appreciate the kind and humble assistance of Prof. Noor Muhammad Butt from Preston University.

Funding

This research has been generously funded through UNSEO-UNISA Africa Chair in Nanosciences and Nanotechnology, and National Research Foundation.

Conflict of interest

All authors declare that they have no conflict of interest.

Author’s contribution

ATK, ZKS, MM for conceiving the idea. ATK, MO, IU for performing the experimental. ATK, MO, MA prepared the draft manuscript. MA, ZKS and MM reviewed and improved the manuscript.

References

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Show Sections

[1] Abamor E.S., . Antileishmanial activities of caffeic acid phenethyl ester loaded PLGA nanoparticles against Leishmania infantum promastigotes and amastigotes in vitro. Asian Pac. J. Trop. Med.. 2017;10(1):25-34.
[Google Scholar]

[2] Adekunle A.S., Pillay J., Ozoemena K.I., . Probing the electrochemical behaviour of SWCNT–cobalt nanoparticles and their electrocatalytic activities towards the detection of nitrite at acidic and physiological pH conditions. Electrochim. Acta. 2010;55(14):4319-4327.
[Google Scholar]

[3] Ai L.-H., Jiang J., . Rapid synthesis of nanocrystalline Co₃O₄ by a microwave-assisted combustion method. Powder Technol.. 2009;195(1):11-14.
[Google Scholar]

[4] Ali A., Ambreen S., Javed R., Tabassum S., ul Haq I., Zia M., . ZnO nanostructure fabrication in different solvents transforms physio-chemical, biological and photodegradable properties. Mater. Sci. Eng. C. 2017;74:137-145.
[Google Scholar]

[5] Askarinejad A., Bagherzadeh M., Morsali A., . Catalytic performance of Mn₃O₄ and Co₃O₄ nanocrystals prepared by sonochemical method in epoxidation of styrene and cyclooctene. Appl. Surf. Sci.. 2010;256(22):6678-6682.
[Google Scholar]

[6] Bregar V.B., Lojk J., Suštar V., Veranič P., Pavlin M., . Visualization of internalization of functionalized cobalt ferrite nanoparticles and their intracellular fate. Int. J. Nanomed.. 2013;8:919-931.
[Google Scholar]

[7] Chung S.-K., Kim Y.-C., Takaya Y., Terashima K., Niwa M., . Novel flavonol glycoside, 7-O-methyl mearnsitrin, from Sageretia theezans and its antioxidant effect. J. Agric. Food Chem.. 2004;52(15):4664-4668.
[Google Scholar]

[8] Contreras J., Xie J., Chen Y.J., Pei H., Zhang G., Fraser C.L., . Intracellular uptake and trafficking of difluoroboron dibenzoylmethane-poly (lactic acid) nanoparticles in HeLa Cells. ACS Nano. 2010;4(5):2735.
[Google Scholar]

[9] Dai G., Liu S., Liang Y., Luo T., . Synthesis and enhanced photoelectrocatalytic activity of p–n junction Co₃O₄/TiO₂ nanotube arrays. Appl. Surf. Sci.. 2013;264:157-161.
[Google Scholar]

[10] de Almeida M.C., Silva A.C., Barral A., Barral Netto M., . A simple method for human peripheral blood monocyte isolation. Mem. Inst. Oswaldo Cruz. 2000;95(2):221-223.
[Google Scholar]

[11] De Faria D., Venâncio Silva S., De Oliveira M., . Raman microspectroscopy of some iron oxides and oxyhydroxides. J. Raman Spectrosc.. 1997;28(11):873-878.
[Google Scholar]

[12] Diallo A., Beye A., Doyle T., Park E., Maaza M., . Green synthesis of Co₃O₄ nanoparticles via Aspalathus linearis: physical properties. Green Chem. Lett. Rev.. 2015;8(3–4):30-36.
[Google Scholar]

[13] Diallo A., Manikandan E., Rajendran V., Maaza M., . Physical & enhanced photocatalytic properties of green synthesized SnO₂ nanoparticles via Aspalathus linearis. J. Alloys Compd.. 2016;681:561-570.
[Google Scholar]

[14] Diallo A., Ngom B., Park E., Maaza M., . Green synthesis of ZnO nanoparticles by Aspalathus linearis: Structural & optical properties. J. Alloys Compd.. 2015;646:425-430.
[Google Scholar]

[15] Dong C., Xiao X., Chen G., Guan H., Wang Y., . Hydrothermal synthesis of Co₃O₄ nanorods on nickel foil. Mater. Lett.. 2014;123:187-190.
[Google Scholar]

[16] Durán N., Marcato P.D., Durán M., Yadav A., Gade A., Rai M., . Mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides, bacteria, fungi, and plants. Appl. Microbiol. Biotechnol.. 2011;90(5):1609-1624.
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[17] Ezhilarasi A.A., Vijaya J.J., Kaviyarasu K., Maaza M., Ayeshamariam A., Kennedy L.J., . Green synthesis of NiO nanoparticles using Moringa oleifera extract and their biomedical applications: cytotoxicity effect of nanoparticles against HT-29 cancer cells. J. Photochem. Photobiol.. 2016;164:352-360.
[Google Scholar]

[18] Farhadi S., Pourzare K., Sadeghinejad S., . Simple preparation of ferromagnetic Co₃O₄ nanoparticles by thermal dissociation of the [Co^II(NH₃)₆](NO₃)₂ complex at low temperature. J. Nanostruct. Chem.. 2013;3(1):16.
[Google Scholar]

[19] Fatima H., Khan K., Zia M., Ur-Rehman T., Mirza B., Haq I.-U., . Extraction optimization of medicinally important metabolites from Datura innoxia Mill.: an in vitro biological and phytochemical investigation. BMC Complement. Altern. Med.. 2015;15(1):376.
[Google Scholar]

[20] Gatselou V., Christodouleas D.C., Kouloumpis A., Gournis D., Giokas D.L., . Determination of phenolic compounds using spectral and color transitions of rhodium nanoparticles. Anal. Chim. Acta. 2016;932:80-87.
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[21] Hadighi R., Mohebali M., Boucher P., Hajjaran H., Khamesipour A., Ouellette M., . Unresponsiveness to Glucantime treatment in Iranian cutaneous leishmaniasis due to drug-resistant Leishmania tropica parasites. PLoS Med.. 2006;3(5):e162.
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[22] Hyun T.K., Song S.C., Song C.-K., Kim J.-S., . Nutritional and nutraceutical characteristics of Sageretia theezans fruit. J. Food Drug Anal.. 2015;23(4):742-749.
[Google Scholar]

[23] Ismail E., Diallo A., Khenfouch M., Dhlamini S., Maaza M., . RuO₂ nanoparticles by a novel green process via Aspalathus linearis natural extract & their water splitting response. J. Alloys Compd.. 2016;662:283-289.
[Google Scholar]

[24] Jafri L., Saleem S., Ullah N., Mirza B., . In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch. Arab. J. Chem.. 2017;10(2):S3699-S3706.
[Google Scholar]

[25] Jakubek T., Kaspera W., Legutko P., Stelmachowski P., Kotarba A., . Surface versus bulk alkali promotion of cobalt-oxide catalyst in soot oxidation. Catal. Commun.. 2015;71:37-41.
[Google Scholar]

[26] Javed R., Usman M., Tabassum S., Zia M., . Effect of capping agents: structural, optical and biological properties of ZnO nanoparticles. Appl. Surf. Sci.. 2016;386:319-326.
[Google Scholar]

[27] Kaviyarasu K., Raja A., Devarajan P.A., . Structural elucidation and spectral characterizations of Co₃O₄ nanoflakes. Spectrochim. Acta Mol. Biomol. Spectrosc.. 2013;114:586-591.
[Google Scholar]

[28] Khan I., AbdElsalam N.M., Fouad H., Tariq A., Ullah R., Adnan M., . Application of ethnobotanical indices on the use of traditional medicines against common diseases. Evid. Based Complement. Alternat. Med.. 2014;2014
[Google Scholar]

[29] Khan I., Ahmad K., Khalil A.T., Khan J., Khan Y.A., Saqib M.S., . Evaluation of antileishmanial, antibacterial and brine shrimp cytotoxic potential of crude methanolic extract of Herb Ocimum basilicum (Lamiacea) J. Tradit. Chin. Med.. 2015;35(3):316-322.
[Google Scholar]

[30] Khan S., Ansari A.A., Khan A.A., Ahmad R., Al-Obaid O., Al-Kattan W., . In vitro evaluation of anticancer and antibacterial activities of cobalt oxide nanoparticles. J. Biol. Inorg. Chem.. 2015;20(8):1319-1326.
[Google Scholar]

[31] Li S., Malmstadt N., . Deformation and poration of lipid bilayer membranes by cationic nanoparticles. Soft Matter. 2013;9(20):4969-4976.
[Google Scholar]

[32] Li W.-Y., Xu L.-N., Chen J., . Co₃O₄ nanomaterials in lithium-ion batteries and gas sensors. Adv. Func. Mater.. 2005;15(5):851-857.
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[33] Limor H.-A., James K.C., Rafi K., Patrice N.M., Oded M., Jessica P., . Predictive toxicology of cobalt nanoparticles and ions: comparative in-vitro study of different cellular models using methods of knowledge discovery from data. Toxicol. Sci. 2011 kfr124
[Google Scholar]

[34] Lin J., Alexander-Katz A., . Cell membranes open “doors” for cationic nanoparticles/biomolecules: insights into uptake kinetics. ACS Nano. 2013;7(12):10799-10808.
[Google Scholar]

[35] Makhlouf M.T., Abu-Zied B., Mansoure T., . Effect of calcination temperature on the H₂O₂ decomposition activity of nano-crystalline Co₃O₄ prepared by combustion method. Appl. Surf. Sci.. 2013;274:45-52.
[Google Scholar]

[36] Malagoli D., . A full-length protocol to test hemolytic activity of palytoxin on human erythrocytes. Inver. Surviv. J.. 2007;4(2):92-94.
[Google Scholar]

[37] Matinise N., Fuku X., Kaviyarasu K., Mayedwa N., Maaza M., . ZnO nanoparticles via Moringa oleifera green synthesis: physical properties & mechanism of formation. Appl. Surf. Sci.. 2017;406:339-347.
[Google Scholar]

[38] Murad W., Ahmad A., Gilani S.A., Khan M.A., . Indigenous knowledge and folk use of medicinal plants by the tribal communities of Hazar Nao Forest, Malakand District, North Pakistan. J. Med. Plants Res.. 2011;5(7):1072-1086.
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[39] Nadhman A., Sirajuddin M., Nazir S., Yasinzai M., . Photo-induced Leishmania DNA degradation by silver-doped zinc oxide nanoparticle: an in-vitro approach. IET Nanobiotechnol.. 2016;10(3):129-133.
[Google Scholar]

[40] Ortega R., Bresson C., Darolles C., Gautier C., Roudeau S., Perrin L., . Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells. Part Fibre Toxicol.. 2014;11(1):14.
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[42] Ovais M., Raza A., Naz S., Islam N.U., Khalil A.T., Ali S., . Current state and prospects of the phytosynthesized colloidal gold nanoparticles and their applications in cancer theranostics. Appl. Microbiol. Biotechnol. 2017:1-15.
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Physical properties, biological applications and biocompatibility studies on biosynthesized single phase cobalt oxide (Co3O4) nanoparticles via Sageretia thea (Osbeck.)

Abstract

Abstract

Keywords

Biosynthesis

Cobalt oxide

Nanoparticles

Antimicrobial

Antileishmanial

Antioxidant

1 Introduction

2 Material and methods

2.1 Plant material processing

2.2 Biosynthesis procedure

2.3 Characterization

2.4 Antibacterial potential

2.5 Brine shrimp cytotoxicity

2.6 Antileishmanial activity (Promastigotes and Amastigotes)

2.7 Biocompatibility assessment

2.7.1 Biocompatibility with macrophages

2.7.2 Hemolytic assay

2.8 Antioxidant activities

2.8.1 DPPH radical scavenging

2.8.2 Reducing power

2.8.3 Total antioxidant capacity

2.9 Enzyme inhibition assays

2.9.1 Protein kinase inhibition

2.9.2 Alpha amylase inhibition

3 Results and discussion

3.1 Biosynthesis of cobalt oxide nanoparticles

3.2 Physical characterization

3.3 Antibacterial activities

3.4 Cytotoxic activities

3.4.1 Brine shrimp cytotoxicity

3.4.2 Antileishmanial activities

3.5 Biocompatibility assessment

3.6 Mechanism of cytotoxicity

3.7 Antioxidant assays

3.8 Enzyme inhibition assays

4 Conclusion

Acknowledgments

Funding

Conflict of interest

Author’s contribution

References

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Physical properties, biological applications and biocompatibility studies on biosynthesized single phase cobalt oxide (Co₃O₄) nanoparticles via Sageretia thea (Osbeck.)