阅读说明：本技术 一种利用多酚类化合物与蛋白质复配制备纳米硒的方法及制成的纳米硒 (Method for preparing nano-selenium by compounding polyphenol compounds and protein and prepared nano-selenium ) 是由 林晓蓉 郑晓峰 李斌 叶锡光 黄欣琪 陈忠正 张媛媛 于 2021-09-16 设计创作，主要内容包括：本发明属于纳米材料技术领域,公开了一种利用多酚类化合物与蛋白质复配制备纳米硒的方法及制成的纳米硒。该方法包括以下步骤：(1)多酚化合物与蛋白质复配为软模板：将一定浓度蛋白质溶液与多酚类化合物溶液混合均匀,直接使用或冻干后使用；(2)利用多酚-蛋白质软模板制备纳米硒：将维生素C溶液加入多酚-蛋白质软模板存在的反应体系中混合均匀,再加入亚硒酸钠溶液搅拌混合均匀,所得混合反应体系静置还原反应得到纳米硒溶胶,透析或离心分离得到纳米硒悬液,冷冻干燥得到纳米硒固体。本发明以多酚类化合物和蛋白质复配为软模板,增强纳米硒功能特性的同时,也增强其稳定性,拓宽纳米硒的应用领域,提高其应用价值。(The invention belongs to the technical field of nano materials, and discloses a method for preparing nano selenium by compounding a polyphenol compound and protein and the prepared nano selenium. The method comprises the following steps: (1) compounding polyphenol compounds and proteins into a soft template: uniformly mixing a protein solution with a certain concentration with a polyphenol compound solution, and directly using or using after freeze-drying; (2) preparing nano selenium by using a polyphenol-protein soft template: adding vitamin C solution into a reaction system in which a polyphenol-protein soft template exists, uniformly mixing, adding sodium selenite solution, uniformly stirring and mixing, standing the obtained mixed reaction system for reduction reaction to obtain nano selenium sol, dialyzing or centrifugally separating to obtain nano selenium suspension, and freeze-drying to obtain nano selenium solid. According to the invention, the polyphenol compound and the protein are compounded into the soft template, so that the functional characteristics of the nano-selenium are enhanced, the stability of the nano-selenium is enhanced, the application field of the nano-selenium is widened, and the application value of the nano-selenium is improved.)

1. A method for preparing nano-selenium by compounding polyphenol compounds and proteins is characterized by comprising the following steps:

(1) compounding polyphenol compounds and proteins to form a polyphenol-protein soft template;

(2) preparing nano selenium by using a polyphenol-protein soft template: adding vitamin C solution into a reaction system in which a polyphenol-protein soft template exists, uniformly mixing, adding sodium selenite solution, uniformly stirring and mixing, standing the obtained mixed reaction system for reduction reaction to obtain nano selenium sol, dialyzing or centrifugally separating to obtain nano selenium suspension, and freeze-drying to obtain nano selenium solid.

2. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 1, which is characterized in that: the polyphenol compounds in the step (1) are compounds with a plurality of phenolic groups, and comprise phenolic acid, flavonoid, stilbene, lignan or procyanidine; the protein is water soluble protein and water insoluble protein, including albumin, globulin, gluten, histone, protamine or scleroprotein.

3. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 2, which is characterized in that: the phenolic acid contains C₆-C₁Or C₆-C₂A carbon chain structure; the lignan contains C₆-C₃A carbon chain structure; said stilbene contains C₆-C₂-C₆A carbon chain structure; the flavonoid and procyanidin contain C₆-C₃-C₆And (4) a carbon chain structure.

4. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 1, which is characterized in that: compounding the polyphenol compound and the protein into the polyphenol-protein soft template in the step (1) specifically comprises the following operations: uniformly mixing a polyphenol compound solution and a protein solution, or adding a polyphenol compound solid into the protein solution for uniform mixing, or adding a protein solid into the polyphenol compound solution for uniform mixing, or mixing the polyphenol compound solid and the protein solid and then adding a solvent for dissolving to finally obtain a solution of a polyphenol-protein soft template; the concentration of the protein solution is 0.1-100 mg/mL; the concentration of the polyphenol compound solution is 0.001-100 mM; the solvents of the protein solution, the polyphenol compound solution, the sodium selenite solution and the vitamin C solution all contain 0-100% of organic solvents.

5. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 4, which is characterized in that: the solution of the polyphenol-protein soft template is directly used for preparing the nano selenium in the step (2), or the solution is frozen and dried to obtain the polyphenol-protein soft template which is stored at the temperature of-20 ℃; the organic reagent is methanol, ethanol, acetone, diethyl ether, ethyl acetate, dimethyl sulfoxide or tetrahydrofuran.

6. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 1, which is characterized in that: the concentration of the sodium selenite solution in the step (2) is 2.5-100 mM, and the concentration of the vitamin C solution is 20-500 mM; the molar ratio of sodium selenite to vitamin C in the mixed reaction system is 1: 2-1: 16; the standing reduction reaction is carried out for 0.5-72 h at the temperature of 4-80 ℃.

7. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 6, which is characterized in that: the concentration of the sodium selenite solution is 100mM, and the concentration of the vitamin C solution is 250 mM.

8. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 1, which is characterized in that: the molar ratio of the sodium selenite to the vitamin C in the mixed reaction system in the step (2) is 1: 8; the concentration of sodium selenite in the mixed reaction system is 5mM, the concentration of protein is 3mg/mL, and the concentration of polyphenol compounds is 3 mM; the standing reduction reaction is carried out for 50min at the temperature of 40 ℃.

9. The method for preparing nano-selenium by compounding the polyphenol compound and the protein according to claim 1, which is characterized in that: dialyzing for 9-96 h by using a 3.5-8 kDa regenerated cellulose dialysis bag; the centrifugation is carried out for 10-30 min at 8000-11000 rpm by adopting a centrifuge.

10. Nano-selenium produced by the method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of nano materials, and particularly relates to a method for preparing nano selenium by compounding a polyphenol compound and protein and the prepared nano selenium.

Background

Selenium (Se) is one of essential trace elements for human body, is a key component of multiple enzyme activity centers in human body, and plays roles of resisting diseases, delaying senility, enhancing immunity and the like in life. Selenium cannot be synthesized by human bodies and can only be supplemented by diet, but the safe dosage range of selenium element is narrow, and excessive intake of selenium element easily causes toxicity. Most of the traditional selenium supplements are inorganic selenium (sodium selenite) and organic selenium (selenomethionine and the like), and researches show that the toxicity of selenium and compounds thereof is as follows: inorganic selenium > organic selenium > elemental selenium. At present, researchers develop amorphous elemental selenium, the particle size of which reaches the nanometer level and is also called nano selenium, and the amorphous elemental selenium has toxicity far lower than that of inorganic selenium and organic selenium under the concentration of generating the same biological effect, so that the amorphous elemental selenium has good application prospect.

The nano-selenium can be generally prepared by methods such as a chemical reduction method, a solvothermal method, a microwave-assisted method, biosynthesis and the like, wherein the chemical reduction method is widely applied due to simple operation and low cost, but the nano-selenium synthesized by the chemical reduction method is easy to agglomerate and loses bioactivity, and needs to be stabilized by an additional template. Recently, many documents report that bioactive substances such as polysaccharide, protein, polyphenol compounds and the like are used as a soft template to stabilize the nano-selenium, wherein the protein soft template has a good effect of stabilizing the nano-selenium, but is difficult to endow other functions to the nano-selenium; although the soft template of the polyphenol compound can endow the nano selenium with additional functions, the stabilizing effect is poor. The method for stabilizing the nano-selenium by compounding the polyphenol compound and the protein into the soft template is not reported so far.

Disclosure of Invention

In order to solve the defects in the prior art, the invention mainly aims to provide a method for preparing nano selenium by compounding polyphenol compounds and proteins.

Another object of the present invention is to provide a nano-selenium prepared by the above preparation method; the nano-selenium has small particle size, narrow particle size distribution range, good dispersibility and good stability.

The purpose of the invention is realized by the following technical scheme:

a method for preparing nano-selenium by compounding polyphenol compounds and proteins comprises the following steps:

(1) compounding polyphenol compounds and proteins to form a polyphenol-protein soft template;

(2) preparing nano selenium by using a polyphenol-protein soft template: adding vitamin C solution into a reaction system in which a polyphenol-protein soft template exists, uniformly mixing, adding sodium selenite solution, uniformly stirring and mixing, standing the obtained mixed system for reduction reaction to obtain nano selenium sol, dialyzing or centrifugally separating to obtain nano selenium suspension, and freeze-drying to obtain nano selenium solid.

The polyphenol compounds in the step (1) are compounds with a plurality of phenolic groups, and comprise phenolic acid, flavonoid, stilbene, lignan or procyanidine; the protein is water soluble protein and water insoluble protein, including albumin, globulin, gluten, histone, protamine or scleroprotein.

Preferably, the phenolic acid contains C₆-C₁Or C₆-C₂A carbon chain structure; the lignan contains C₆-C₃A carbon chain structure; said stilbene contains C₆-C₂-C₆A carbon chain structure; the flavonoid and procyanidin contain C₆-C₃-C₆And (4) a carbon chain structure.

Compounding the polyphenol compound and the protein into the polyphenol-protein soft template in the step (1) specifically comprises the following operations: uniformly mixing a polyphenol compound solution and a protein solution, or adding a polyphenol compound into the protein solution, or adding a protein solid into the polyphenol compound solution, or uniformly mixing, or mixing the polyphenol compound solid with the protein solid, and then adding a solvent for dissolving to finally obtain a solution of a polyphenol-protein soft template; the concentration of the protein solution is 0.1-100 mg/mL, and the concentration of the polyphenol compound solution is 0.001-100 mM; the solvents of the protein solution, the polyphenol compound solution, the sodium selenite solution and the vitamin C solution all contain 0-100% of organic solvents.

The solution of the polyphenol-protein soft template is directly used for preparing the nano selenium in the step (2), or the solution is frozen and dried to obtain the polyphenol-protein soft template which is stored at the temperature of-20 ℃; the organic reagent is methanol, ethanol, acetone, diethyl ether, ethyl acetate, dimethyl sulfoxide or tetrahydrofuran.

The concentration of the sodium selenite solution in the step (2) is 2.5-100 mM, and the concentration of the vitamin C solution is 20-500 mM; the molar ratio of sodium selenite to vitamin C in the mixed reaction system is 1: 2-1: 16; the standing reduction reaction is carried out for 0.5-72 h at the temperature of 4-80 ℃.

The concentration of the sodium selenite solution is 100mM, and the concentration of the vitamin C solution is 250 mM.

The molar ratio of the sodium selenite to the vitamin C in the mixed reaction system in the step (2) is 1: 8; the concentration of sodium selenite in the mixed reaction system is 5mM, the concentration of protein is 3mg/mL, and the concentration of polyphenol compounds is 3 mM; the standing reduction reaction is carried out for 50min at the temperature of 40 ℃.

Dialyzing for 9-96 h by using a 3.5-8 kDa regenerated cellulose dialysis bag; the centrifugation is carried out for 10-30 min at 8000-11000 rpm by adopting a centrifuge.

Nano-selenium prepared according to the method.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the invention adopts the compounding of polyphenol compounds and protein as a soft template; the polyphenol compounds have antioxidant, anticancer, antiinflammatory, blood lipid reducing, weight reducing and neuroprotective effects; the polyphenol compounds and the protein can form a compound through hydrogen bond interaction and hydrophobic interaction, and then are combined to the surface of the nano selenium through the two acting forces to fully disperse and stabilize the nano selenium; the soft template compounded by the polyphenol compounds and the protein endows the nano selenium with good stability and has the health effect of the polyphenol compounds, thereby achieving the synergistic effect.

(2) The physiological activity of protein is rarely reported, and after the polyphenol substances are added, the polyphenol substances have a stabilizing effect and also have additional physiological activity, so that the polyphenol substances are required to be ensured not to be damaged in the preparation process; after the polyphenol compounds and the proteins form a compound, firstly adding vitamin C to change the pH of the solution into acidity and simultaneously protect the polyphenol compounds from being oxidized, and then adding sodium selenite; if sodium selenite is added firstly, the polyphenol substances are unstable, the structure and the function of the polyphenol substances are damaged, and the polyphenol substances and the protein can be combined in a covalent bond mode, and the adding sequence of the invention can protect the structure of the polyphenol substances to the maximum extent and can be combined with the protein in a non-covalent mode under the acidic condition.

Drawings

FIG. 1 is a graph showing the effect of different sodium selenite concentrations on the nano-selenium particle size;

FIG. 2 is a graph showing the effect of different ratios of sodium selenite to vitamin C on the particle size of nano-selenium;

FIG. 3 is a graph showing the effect of different BSA concentrations on the particle size of nano-selenium;

FIG. 4 is a graph showing the effect of different EGCG concentrations on the nano-selenium particle size;

FIG. 5 is a graph showing the effect of different reaction temperatures on the particle size of nano-selenium;

FIG. 6 is a graph showing the particle sizes of chlorogenic acid-BSA nano-selenium and ferulic acid-BSA nano-selenium;

FIG. 7 is a graph showing the effect of different orders of addition of reactants on the particle size of nano-selenium;

FIG. 8 is a graph of endogenous fluorescence quenching of BSA with different concentrations of EGCG;

FIG. 9 is a plot of fitted lines of the Stern-Volmer equation at different reaction temperatures;

FIG. 10 is a graph of a log-log fit line at different reaction temperatures.

Detailed description of the invention

The present invention will be described in further detail below with bovine serum albumin BSA and the polyphenol compound epigallocatechin gallate (EGCG), chlorogenic acid and ferulic acid as examples and figures, and the equipment and reagents used are all commonly used in the art, but the embodiments of the present invention are not limited thereto.

Example 1: method for preparing nano-selenium by compounding polyphenol compounds and protein

1) 100mM sodium selenite solution, 250mM vitamin C solution, 10mg/mL BSA solution and 10mM EGCG solution are prepared respectively.

2) Fixing the concentration of BSA in the final mixed reaction system to be 5mg/mL and the concentration of EGCG to be 3mM, and firstly, uniformly mixing a certain amount of BSA solution and EGCG solution to obtain a reaction system; fixing the molar ratio of sodium selenite to vitamin C to be 10:1, respectively adding a certain amount of vitamin C solution into a reaction system according to the concentration of sodium selenite in the final mixed reaction system of 2.5, 5, 7.5, 10, 12.5 and 15mM, uniformly mixing, adding the sodium selenite solution, stirring for 10min, uniformly mixing, standing at 25 ℃ for 50min, and obtaining a nano-selenium colloid solution;

3) and dialyzing the nano selenium colloidal solution for 24 hours by using an 8kDa regenerated cellulose dialysis bag, removing unreacted substances, and determining the chemical characteristics of the colloid, wherein the results are shown in figure 1, and comprehensively considered, the average particle size is smaller when the concentration of sodium selenite in the mixed reaction system is 5mM, the nano selenium suspension is clearer and brighter, and the system is more stable.

4) The dialyzed nano selenium colloidal solution is frozen and dried, and then is stored at the temperature of minus 20 ℃ for standby.

Example 2: method for preparing nano-selenium by compounding polyphenol compounds and protein

Compared with example 1, the difference is that:

preparing 500mM vitamin C solution, fixing the concentration of sodium selenite in the final mixed reaction system to be 5mM, adding a certain amount of 500mM vitamin C solution according to the molar ratio of sodium selenite to vitamin C of 1:4, 1:6, 1:8, 1:10, 1:12 and 1:16(mmol: mmol), mixing uniformly, adding 100mM sodium selenite solution, stirring for 10min, mixing uniformly, standing for 50min at 25 ℃ to obtain nano-selenium colloidal solution, and measuring the chemical characteristics of the colloid, wherein the results are shown in figure 2.

Example 3: method for preparing nano-selenium by compounding polyphenol compounds and protein

Compared with example 1, the difference is that:

preparing 30mg/mL BSA solution, uniformly mixing a certain amount of 30mg/mL BSA solution with 10mM EGCG solution according to the respective concentration of 1, 3, 5, 7 and 9mg/mL BSA in the final mixed reaction system, fixing the concentration of sodium selenite in the final mixed reaction system to be 5mM, firstly adding a certain amount of 250mM vitamin C solution according to the molar ratio of sodium selenite to vitamin C of 1:8(mmol: mmol), uniformly mixing, then adding a certain amount of 100mM sodium selenite solution, stirring for 10min, uniformly mixing, standing for 50min at 25 ℃ to obtain a nano-selenium colloid solution, and measuring the colloid chemical characteristics of the nano-selenium colloid solution.

Example 4: method for preparing nano-selenium by compounding polyphenol compounds and protein

Compared with example 1, the difference is that:

fixing the concentration of BSA in the final mixed reaction system to be 3mg/mL and the concentration of EGCG to be 1, 2, 3, 4 and 5mM respectively, uniformly mixing a certain amount of 10mg/mL BSA solution with a 20mM EGCG solution, fixing the concentration of sodium selenite in the final mixed reaction system to be 5mM, firstly adding a certain amount of 250mM vitamin C solution according to the molar ratio of sodium selenite to vitamin C being 1:8(mmol: mmol), uniformly mixing, then adding a certain amount of 100mM sodium selenite solution, stirring for 10min, uniformly mixing, standing for 50min at 25 ℃ to obtain a nano-selenium colloid solution, and measuring the colloid chemical characteristics of the nano-selenium colloid solution.

Example 5: method for preparing nano-selenium by compounding polyphenol compounds and protein

Compared with example 1, the difference is that:

fixing the concentration of BSA in the final mixed reaction system to be 3mg/mL, uniformly mixing a certain amount of 10mg/mL BSA solution with a 10mM EGCG solution, fixing the concentration of sodium selenite in the mixed reaction system to be 5mM, firstly adding a certain amount of 250mM vitamin C solution according to the molar ratio of sodium selenite to vitamin C being 1:8(mmol: mmol), uniformly mixing, then adding a certain amount of 100mM sodium selenite solution, stirring for 10min, uniformly mixing, respectively standing for 4, 25, 40 and 60 ℃ for reaction for 50min to obtain a nano-selenium colloidal solution, and measuring the chemical characteristics of the nano-selenium colloidal solution.

Example 6: method for preparing nano-selenium by compounding polyphenol compounds and protein

Compared with example 1, the difference is that:

fixing the concentration of BSA in the final mixed reaction system to be 3mg/mL, uniformly mixing a certain amount of 10mg/mL BSA solution with 10mM chlorogenic acid or ferulic acid solution, wherein the concentration of sodium selenite in the fixed system is 5mM, firstly adding a certain amount of 250mM vitamin C solution according to the ratio of 1:8(mmol: mmol) of sodium selenite to vitamin C, uniformly mixing, adding a certain amount of 100mM sodium selenite solution, stirring for 10min, uniformly mixing, standing for 50min at 40 ℃ for reaction to obtain a nano-selenium colloidal solution, dialyzing the nano-selenium colloidal solution for 9h by using an 8kDa regenerated cellulose dialysis bag, removing unreacted substances, and determining the colloidal chemical characteristics, wherein the result is shown in figure 6.

Example 7: method for preparing nano-selenium by compounding polyphenol compounds and protein

Compared with example 1, the difference is that:

in the final mixed reaction system, the concentration of immobilized BSA is 3mg/mL, the concentration of immobilized sodium selenite is 5mM, the ratio of immobilized sodium selenite to vitamin C is 1:8(mmol: mmol), and the concentration of immobilized EGCG is 3 mM; the nano selenium colloidal solution is dialyzed for 24h by 8kDa regenerated cellulose dialysis bags respectively, unreacted substances are removed, the chemical characteristics of the nano selenium colloidal solution are measured, and the result is shown in figure 7. The detailed addition sequence is as follows:

uniformly mixing a certain amount of 10mg/mL BSA solution with a certain amount of 10mM EGCG solution, adding a certain amount of 100mM sodium selenite solution, uniformly mixing, finally adding a certain amount of 250mM vitamin C solution, uniformly mixing by stirring for 10min, and standing for reacting for 50min at 40 ℃;

uniformly mixing a certain amount of 10mg/mL BSA solution with 100mM sodium selenite solution, adding a certain amount of 250mM vitamin C solution, uniformly mixing, finally adding a certain amount of 10mM EGCG solution, stirring for 10min, uniformly mixing, standing for 50min at 40 ℃, and reacting for 50 min;

③ evenly mixing a certain amount of 10mg/mL BSA solution with 250mM vitamin C solution, adding a certain amount of 10mM EGCG solution, evenly mixing, finally adding a certain amount of 100mM sodium selenite solution, stirring for 10min, evenly mixing, standing for 50min at 40 ℃;

fourthly, uniformly mixing a certain amount of 10mg/mL BSA solution with a 250mM vitamin C solution, adding a certain amount of 100mM sodium selenite solution, uniformly mixing, finally adding a certain amount of 10mM EGCG solution, stirring for 10min, uniformly mixing, standing for 50min at 40 ℃, and reacting for 50 min;

fifthly, uniformly mixing a certain amount of 10mg/mL BSA solution with a 10mM EGCG solution, adding a certain amount of 250mM vitamin C solution, uniformly mixing, finally adding a certain amount of 100mM sodium selenite solution, stirring for 10min, uniformly mixing, and standing for 50min at 40 ℃.

Example 8: method for preparing nano-selenium by compounding polyphenol compounds and protein

Firstly, preparing a BSA solution with the concentration of 3mg/mL and a 50 mu M EGCG solution, firstly adding 0.5mL BSA solution, then adding a proper amount of EGCG solution, enabling the final concentration of the solution EGCG to be 0, 0.5, 1, 1.5, 2, 2.5, 3 and 3.5 mu M, finally adding a proper amount of primary water until the volume of the solution is 3mL, uniformly mixing, then placing the solution EGCG in a 298K (25 ℃) water bath for heating for 1h in a dark place, respectively measuring the fluorescence intensity of the solution at 280 and 450nm under the excitation wavelength of 280nm, and obtaining a fluorescence quenching curve graph of the EGCG to the BSA, wherein the fluorescence quenching curve graph is shown in figure 8.

② the above experiment is carried out under 303K (30 ℃), and then Stern-Volmer equation F₀/F＝1+K_qτ₀Q＝1+K_svQ denotes the type of fluorescence quenching, wherein₀For the average fluorescence lifetime of fluorescent molecules in the absence of a quencher, it is generally about 10 for most biomacromolecules such as BSA^-8s, Q is quencher concentration mol.L^-1，F₀The endogenous fluorescence intensity of BSA without addition of a quencher, F the endogenous fluorescence intensity of BSA after addition of a quencher, K_qIs a quenching rate constant when it is greater than the maximum diffusion collision quenching constant of 2X 10¹⁰L·mol^-1·s^-1And as the temperature increases, the Stern-Volmer constant K_svThe decrease indicates that the quenching mechanism is static quenching, i.e., complex formation, rather than collision-induced energy transfer leading to fluorescence quenching. As shown in fig. 9 and the results in table 1, it was shown that EGCG and BSA form a complex.

③ after determining to be static quenching, passing through log-log equation lg [ (F)₀-F)/F]＝lgK_a+ nlgQ determination of the binding constant K_aThen through lnK_aThe type of binding force between polyphenol and protein is determined by the equation ═ - (Δ H/RT) + (Δ S/R) and Δ G ═ Δ H-T Δ S, where R is the gas constant, typically 8.314J/(mol · K) and T is the temperature (K). Typical hydrophobic forces are Δ S > 0 and Δ H > 0; Δ H < 0, Δ S < 0 are primarily present with hydrogen bonding and van der Waals forces; when Δ H < 0 and Δ S > 0, electrostatic interaction mainly occurs. The resulting log-log fitted line graph is shown in FIG. 10, where the intercept of the linear equation is lgK_a. The results of the calculations shown in Table 2, where Δ H < 0 and Δ S < 0, can determine that the interaction forces of EGCG with BSA are mainly hydrogen bonds and van der Waals forces, and Δ G < 0, indicating that the process is spontaneous.

TABLE 1 Stern-Volmer and quenching constants for the EGCG-BSA system

TABLE 2 Ka, Δ H, Δ S and Δ G for EGCG-BSA system

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.