2016 Volume 63 Issue 7 Pages 668-674
Calcium oxide and small amount of alumina powders were mechanically milled in a planetary ball milling system using ZrO2 pot and balls, in order to refine the sizes of the powders and promote the reactions between the raw powders. The obtained powders were subjected to an accelerated degradation test in CO2/H2O atmosphere, and the antiviral activity was evaluated by using an avian influenza virus strain (H5N3). The mechanically milled powders revealed a significant improvement in durability of antiviral activity after the powders were hydrated. One of the reasons seems to be associated with the solid solution of Al2O3 into calcium oxide and agglomeration of the powders during the mechanical milling. Moreover, it was found that ZrO2 has an effect similar to Al2O3. The incorporation of ZrO2 in CaO by either addition of ZrO2 powder or contamination from milling media can also improve the durability of antiviral activity of CaO.
In recent years, frequent outbreaks of human and highly pathogenic avian influenza have resulted in big economical and social threats all over the world. One of the important preventive measures to reduce spread of the viruses is to develop novel antiviral materials. Recently, dolomite (CaMg(CO3)2) has attracted much attention as one of the candidate antiviral materials. It was reported that heated dolomite powders exhibited antibacterial activities1), and heated and hydrated dolomite has not only antibacterial activity, but also antiviral activity against human and avian influenza viruses as well as against avian infectious bronchitis2–3). It is believed that the antiviral activity of the processed dolomite is associated with hydration of calcium oxide (CaO)4–5). Although the processed dolomite has faster-acting and higher antiviral activity in comparison with other inorganic antibacterial materials, its effectiveness of antiviral activity is very limited. This is because hydrated CaO (i.e., Ca(OH)2) easily reacts with CO2 in air and changes into CaCO3, thus resulting in disappearance of antiviral activity.
Several metallic oxides, such as CaO and MgO, have been found to have good antimicrobial activity6–9). Unfortunately, they also have a durability problem, just like dolomite. To improve the durability of antiviral materials, we have developed a CaO-based antiviral material, in which CaO is a main component with small amount of Al2O3 addition10). This material has been prepared by a conventional powder metallurgy route including powder compaction and sintering. The effects of alumina addition and some processing conditions (such as compaction pressure and sintering temperature) on antiviral activities of CaO have been investigated in the previous paper10). In the present work, instead of sintering process, a mechanical milling process has been proposed to synthesize CaO-based antiviral material. The purpose was to clarify the effect of mechanical milling on phase constitution, microstructure, and antiviral activity.
CaO and γ-Al2O3 powders were used as the starting materials in the experiments. The raw powders with nominal compositions of CaO-0, 1, 5, 10 % Al2O3 were ball-milled in ethanol for 24 h. After drying, the powder mixtures were further treated by mechanical milling in a planetary ball milling system under different milling conditions. The mechanical milling was conducted using ZrO2 pot and balls under 300 rpm, and ball to powder weight ratio was fixed at 50:1. The mechanically milled (MMed) powders were hydrated to change CaO into Ca(OH)2.
In order to evaluate the degradation behavior, the MMed powders were subjected to an accelerated degradation test in CO2/H2O atmosphere at 40 °C for 24 h. The crystalline phases and lattice constants were determined by X-ray diffraction (XRD) with CuKα radiation. Microstructural characterization was performed by SEM and TEM, and energy dispersive X-ray spectroscopy (EDS) was used for compositional analysis with TEM. In addition, some powder samples were diluted with phosphate buffered saline (PBS) to a concentration of 5 % and their pH values were measured by a pH meter.
The antiviral activity was examined by using the avian influenza virus strain A/whistiling swan/Shimane/499/83 (H5N3). The strain had been grown in the allantoic cavity of 10-day-old embryonated specific pathogen-free (SPF) hen’s eggs for 3 days at 37 °C prior to the test. The strength of the antiviral activity was measured by the degree of dilution which resulted in an infection of 50 % of the objects. The 50 % egg infectious dose (EID50) was calculated by the method of Reed and Muench11).
In order to identify the crystalline phases in MMed powders and hydrated samples, XRD analysis has been performed. As an example, Fig. 1 shows the XRD patterns of the CaO powders with and without mechanical milling and/or hydration. The peaks in the XRD pattern of the MMed powder (Fig. 1 (b)) were lower and broader compared to the non-milled powder (Fig. 1 (a)). The broadened peaks are believed to result from small crystallite sizes and lattice strain, which are introduced during the milling process. When the MMed and non-MMed powders were hydrated, as expected, CaO was changed into Ca(OH)2. As a result, strong Ca(OH)2 peaks appeared in both MMed and non-MMed powders (Figs. 1 (d) and (c)). In addition, small peaks of CaCO3 were observed in the hydrated powders, due to reaction between Ca(OH)2 and CO2 in the atmosphere.
XRD profiles of CaO powders (a) without mechanical milling, (b) with mechanical milling for 8h, (c) after hydration for non-milled powder, and (d) after hydration for 8h milled powder.
In order to evaluate the durability, the hydrated powders were subjected to an accelerated degradation test. Fig. 2 (a) shows the XRD patterns of hydrated and then degraded CaO samples (CaO-0 %Al2O3) with different milling times. The peaks of CaCO3 were observed in all the patterns, as shown in Fig. 2 (a), indicating that carbonation reactions occur during the degradation tests. For the powders MMed for ≤ 4 h, all the peaks correspond to CaCO3. With regard to the sample MMed for 8 h, however, small but well-defined peaks of Ca(OH)2 remained in addition to CaCO3. These results suggest that mechanical milling is beneficial to delay the carbonation reactions and improve the durability of hydrated powder (Ca(OH)2).
XRD profiles of hydrated CaO-Al2O3 powders with different milling times after degradation tests.
For the purpose of further improving the durability of antiviral activity, small amounts of Al2O3 powder were incorporated into CaO. Powder mixtures with compositions of CaO-(1–10) % Al2O3 were mechanically milled for different times, followed by hydration and degradation tests, just like the treatment of CaO without Al2O3 addition as stated above. Figs. 2 (b)–(d) illustrate the XRD patterns of CaO-Al2O3 samples after the degradation tests. Although the main phase is CaCO3 in all the samples after the degradation tests, as shown in Figs. 2 (b)–(d), Ca(OH)2 phase is present in the samples of CaO-1 % Al2O3 MMed for ≥ 1 h, CaO-5 % Al2O3 MMed for ≥ 0.5 h, and CaO-10 % Al2O3 MMed for ≥ 0.5 h, respectively. In comparison with Fig. 2 (a), it is obvious that the addition of Al2O3 results in shortening of milling time needed for remaining of Ca(OH)2 phase after the degradation tests. This indicates that the incorporation of Al2O3 in CaO may promote the improvement of durability of antiviral activity, similar to the sintered CaO-Al2O3 samples10).
It is widely recognized that strong alkaline substance is responsible for strong antiviral activity. Accordingly, pH value is to some extent a simple measure to evaluate antiviral activity. In the present work, prior to the degradation tests, all the samples, including those without Al2O3 addition and MM treatment, showed strong alkaline with pH values of > 12, because the dominant phase is Ca(OH)2. After the degradation tests, the pH test results of samples are shown in Fig. 3. The non-mechanically milled samples, indicated as 0 h, had lower pH values. The CaO-0 % Al2O3 powders MMed for 1 h and 8 h exhibited pH values over 12, revealing a strong alkaline feature. All the MMed samples of CaO with 1 % and 5 % Al2O3 as well as CaO-10 % Al2O3 samples MMed for ≥ 1 h exhibited pH values over 12. These results are in good agreement with the XRD results shown in Fig. 2.
Comparison of pH values of CaO-Al2O3 powders after degradation tests.
Fig. 4 shows the variation of antiviral activity of the samples with and without Al2O3 addition after the degradation tests. All the non-mechanically milled samples had high EID50 values, while all the MMed samples with and without Al2O3 showed much lower EID50 values (that is, better antiviral activity) when the milling time is ≥ 1 h. Moreover, CaO-5 %Al2O3 sample also showed a lower EID50 value, even though mechanical milling time was 0.5 h. These results are in good agreement with the pH results as shown in Fig. 3. Therefore, for Al2O3-containing samples, the effect of mechanical milling is similar to sintering10) and milled powders indicate an excellent antiviral activity. This might be duo to the formation of CaO-Al2O3 solid solution during mechanically milling, like high temperature sintering. On the other hand, CaO powders without Al2O3 addition showed low EID50 values and thus excellent antiviral activity when they were mechanically milled for more than 1 h.
Variation of antiviral activity of CaO-Al2O3 powders after degradation tests.
Fig. 5 shows the change of lattice constant of CaO with milling time. The lattice constant exhibited an increase tendency with increasing milling time. This might be attributed to the formation of CaO-Al2O3 solid solution during mechanical milling. However, the lattice constant of CaO without Al2O3 addition also increases with milling time. EPMA analysis has confirmed that Zr element was detected in the MMed CaO powder. Since ZrO2 pot and balls were used in mechanical milling in the current experiments, it is reasonable to consider that the presence of Zr in the MMed CaO powder should result from abrasion of ZrO2 milling media. Zr element seems to be solid-soluted into CaO during the mechanical milling, thus resulting in formation of CaO-ZrO2 solid solution and improvement of antiviral activity. To examine this possibility, CaO-1 %ZrO2 powders were mechanically milled for different times, followed by hydration and degradation tests. As shown in Fig. 6 (a), Ca(OH)2 phase exists in the powders MMed for ≥ 2 h after the degradation tests. The powders MMed for ≥ 2 h also exhibited pH values higher than 12 and much lower EID50 values, as shown in Figs. 6 (b) and (c), respectively. Accordingly, addition of small amount of ZrO2 can also improve the durability of antiviral activity of CaO.
Dependence of lattice constant of CaO-Al2O3 samples on mechanical milling time.
(a) XRD profiles, (b) comparison of pH values, and (c) antiviral activity of CaO-1 % ZrO2 powders with different milling times after degradation tests.
It was reported that the presence of tricalcium aluminate (3CaO·Al2O3, denoted as C3A) improved the durability of antiviral activity10). In fact, small amount of C3A phase was found in CaO-10 % Al2O3 powders after mechanically milling. To examine the effect of C3A, in the present study, C3A was synthesized from CaO and Al2O3 by solid reactions at a high temperature, and then C3A and CaO powders were mixed with a nominal composition of CaO-1 % Al2O3. The mixed powder was mechanically milled for different times, followed by hydration and degradation tests. After the degradation tests, Ca(OH)2 phase was not found in XRD profiles of the powder. Furthermore, the powder exhibited a low pH value of less than 12 and high EID50 value of about 4.5, indicating that CaO-1 % Al2O3 powder made from CaO and C3A does not have the durability of antiviral activity. Consequently, the presence of C3A in the sintered sample revealed improvement in durability of antiviral activity, however, in the mechanically milled sample, the use of C3A as a starting material is not beneficial to improvement in durability of antiviral activity.
From SEM observations, the agglomerations of particles were confirmed in the MMed powders, as shown in Fig. 7. TEM observations showed a lot of small Al2O3 particles around CaO particles in non-mechanically milled samples, while small Al peak was detected in the MMed powders, as shown in Figs. 8 (a) and (c). Besides, rod-like grains were also observed, as shown in Fig. 8 (b). Fig. 8 (d) shows the EDS spectrum of the rod-like grain in Fig. 8 (b) showing Al peak clearly. The agglomerations of particles during milling process may give rise to decrease in contact areas of Ca(OH)2 with surrounding CO2, thus reducing degradation speed and improving the durability of antiviral activity.
SEM images of hydrated CaO-Al2O3 powders. (a) CaO-0 % Al2O3, MM0h, (b) CaO-0 % Al2O3, MM8h, (c) CaO-1 % Al2O3, MM0h, and (d) CaO-1 % Al2O3, MM8h.
(a) and (b) TEM images of hydrated CaO-10 % Al2O3 powders MMed for 8h. (c) and (d) EDS spectra of grain shown in (a) and rod-like particle in (b), respectively.
This work was supported in part by JSPS KAKENHI Grant Number 26630317 and Kieikai Research Foundation.
