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Table of Contents
ORIGINAL ARTICLE
Year : 2018  |  Volume : 4  |  Issue : 2  |  Page : 54-61

Using the Box–Behnken response surface method to optimize the preparation and characterization of lavender and fennel volatile oil β-cyclodextrin form inclusion complex


1 The National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Jiangxi, China
2 Chinese Medicine Research Institute, Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences, Dongzhimen, Dongcheng District, Beijing 100700, China
3 Basic Medical College, Xinjiang Medical University, Xinjiang, Wulumuqi 830011, China

Date of Web Publication2-Jul-2018

Correspondence Address:
Dong-Xun Li
The National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, 56 Yangming Road, Jiangxi, Nanchang 330006
China
Guo-Song Zhang
The National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, 56 Yangming Road, Jiangxi, Nanchang 330006
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_9_18

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  Abstract 


Objective: The objective of this study is to study the best inclusion technology of Lavender- and fennel-mixed volatile oil by beta-cyclodextrin (β-CD) and characterize the final product thereafter. Methods: Using the saturated water solution method, the volatile β-CD inclusion complex was produced. The effect of volatile oil weight ratio, inclusion temperature and inclusion time on the inclusive quality was studied by measuring the yield of inclusion and inclusion rate of volatile oil as evaluation indexes. The preparation method of inclusion complex was then optimized by the Box-Behnken response surface method. The inclusion complex was characterized by ultraviolet spectrophotometry, thin-layer chromatography, thermogravimetry and differential thermal analysis, and the microscopic imaging method. Results: The optimized conditions were the weight ratio of β-CD to volatile oil was 8.13:1 (g/ml). The inclusion temperature was 44°C. The inclusion time was 1 h. Conclusion: We were able to produce an inclusion complex with high inclusion rate of volatile oil and high yield of inclusion using the preparation method mentioned above. Furthermore, the method can also improve the stability of volatile oil in abnormal savda munziq. This study can provide a good reference for the development of new preparations.

Keywords: Fennel, inclusion complex, lavender, response surface method, volatile oil, β-cyclodextrin


How to cite this article:
Liu YY, Zhang GS, Zhu GW, Aikemu AN, Ai JJ, Li DX. Using the Box–Behnken response surface method to optimize the preparation and characterization of lavender and fennel volatile oil β-cyclodextrin form inclusion complex. World J Tradit Chin Med 2018;4:54-61

How to cite this URL:
Liu YY, Zhang GS, Zhu GW, Aikemu AN, Ai JJ, Li DX. Using the Box–Behnken response surface method to optimize the preparation and characterization of lavender and fennel volatile oil β-cyclodextrin form inclusion complex. World J Tradit Chin Med [serial online] 2018 [cited 2018 Jul 20];4:54-61. Available from: http://www.wjtcm.net/text.asp?2018/4/2/54/235829




  Introduction Top


Lavender belongs to the labiatae plants. When dry, it has strong aroma. The essential oil refined from lavender has good quality and fragrance. The oil, also known as the “king of the spices,” can promote blood circulation, enhance immunity, and increase functional activity, among many other benefits.[1] The main component is volatile oil, which has antibacterial and antioxidant effects,[2] is used as treatment for hypertension,[3] can induce sedation hypnosis [4] and is a neuroprotector.[5],[6] Fennel, an umbrella plant, is used in uyghur medicine, and its active ingredient, Fennel has antibacterial and anti-inflammatory effects and is used as analgesic as well as to treat flooding.[7] Lavender and fennel are important ingredients in classic prescriptions of abnormal savda munziq (ASMq).[8],[9] The volatile oil, used as the main active ingredient, has unstable characteristics. Thus, solving the problems of the stability of volatile oil in the ASMq is critical. Therefore, this study used the technology of beta-cyclodextrin (β-CD) inclusions, together with the Box–Behnken response surface method to optimize the technology, thus obtaining the best preparation for ASMq. Various analyses were conducted to determine the characteristics of ASMq, so as to verify that the inclusion process is reasonable and feasible. This study enhances the stability of lavender- and fennel-mixed volatile oil in the preparation, thus ensuring the efficacy of the drug, which lays the foundation for the development of safe, effective, and stable modern Chinese Medicine Preparations.

Materials and reagents

Lavender was obtained from XinJiang Maddison Weiyao Pharmaceutical Co., Ltd. Fennel was obtained from JiangXi JiangMiddle Chinese Medicine YinPian co., Ltd. β-CD was purchased from TianJin DeMao Chemical Reagent Factory. The remaining reagents used in this study were analytical grade.


  Methods Top


Preparation of mixed volatile oil of lavender and fennel

According to the proportion of the ASMq prescription, the weighed lavender and fennel, at the most coarse powder of 150 g, were added to water at a ratio of 1:10. The volatile oil was extracted by steam distillation during 6 h. The lavender and fennel volatile oils were collected and sealed for preservation.

Determination of the relative density of volatile oil

The pycnometer method, referred from the “Chinese Pharmacopoeia” 2015 edition of the four General Rules 0601 “relative density determination method”[10] was used to calculate the relative density of the volatile oil. The calculation formula is described as follows:

The relative density of the test product = weight of the test product/weight of water

Determination of volatile oil blank recovery

The A method, referred from the “Chinese Pharmacopoeia” 2015 edition of the four General Rules 2204 “Determination method of volatile oil”[11] was used to calculate the volatile oil blank recovery. The calculation formula is described as follows:

Blank recovery = recoveries of volatile oil (ml)/the amount of volatile oil input (mL) × 100%

Inclusion rate of volatile oil and yield of inclusion complex

The volatile oil drying inclusion complex was accurately weighted and placed inside a round-bottomed flask. Then, 300 mL of purified water were added and mixed evenly, in accordance with the method referred in 3.1, to extract and collect volatile oil. While accurately reading the oil content (mL) of the inclusion complex, the inclusion rate of volatile oil was calculated. Using the dried inclusion complex weight of the finished product, the yield of the inclusion complex was calculated. The calculation formula is described as follows:

Inclusion rate of volatile oil = the actual volatile oil content in inclusion complex/(the amount of volatile oil input × blank recovery) × 100%

Yield of inclusion complex = inclusion complex actual amount/(β-CD amount + the amount of volatile oil input × volatile oil relative density) × 100%

Determination of comprehensive evaluation indexes

The experiment was conducted using the inclusion rate of the volatile oil, and the yield of the inclusion complex as an index. These indexes were given appropriate weight coefficients, which led to a comprehensive score regarding the optimal inclusion process. The inclusion rate of the volatile oil is an important index to measure the inclusion effect. The higher the inclusion rate is, the better the inclusion effect of the volatile oil is. Therefore, the weight coefficient assigned to the inclusion rate of the volatile oil, being the main index, was 0.7. The yield of the inclusion complex also occupies an important position in the industry actual production application, that is, in the same amount of the β-CD, the value of the yield of inclusion complex value is greater, which proves that the inclusion effect of the volatile oil is better. Therefore, its weight coefficient was set to 0.3. The calculation formula is described as follows:

Comprehensive score value = ([(inclusion rate of volatile oil)/(the maximum inclusion rate of volatile oil)] × 0.7 + [(yield of inclusion complex)/(the maximum yield of inclusion complex)] × 0.3) × 100

Research on the preparation process of inclusion complex

A certain amount of distilled water was taken into a plug conical bottle of 250 ml with a proper amount of β-CD, thus making a saturated solution of β-CD. Afterward, the mixture of volatile oil and anhydrous ethanol solution (volatile oil: Anhydrous ethanol = 1:1), prepared under the “3.1” item, was slowly added to the saturated β-CD solution with a certain temperature (It shows 30°C, 45°C, and 60°C) while stirring. Then, we continued stirring with the blender until the prescribed time (It shows 0.5 h, 1.0 h and 1.5 h). The sample was removed and naturally cooled down to room temperature and later preserved in the refrigerator at 4°C for 24 h. Then, the sample was quickly filtered and rinsed with a small amount of purified water at first, and then, washed three times with petroleum ether at 60°C–90°C, each time with 10 ml to remove the unfolded volatile oil. Then, the sample was filtered to dry out, in conditions of 40°C for 4 h. Finally, the inclusion complex product was removed and obtained.

Design of the inclusion process test

The level of factors was determined by referring to the literature and at the early stage of the single-factor experiments, to determine the β-CD: volatile oil volume, inclusion temperature (B), and inclusion time (C) as the main factors affecting the β-CD inclusion volatile oil process. Three levels were set for each factor, using the response surface method optimization, as shown in [Table 1].
Table 1: Response surface factor and level

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Experiment verification

In accordance with the above optimal parameters, the mixture of lavender and fennel mixed with volatile oil was carried out in three parallel groups. The results are shown in [Table 2].
Table 2: Results of inclusion verification test

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Characterization of the inclusion complexes

Thin layer chromatography

An amount of volatile oil was transferred accurately to 10 μl. Then, 50 ml of petroleum ether were added, shaking well, and keeping static. Afterward, 2 ml of the top cleaning fluid were pipetted, oil ether was added to make 30 ml, and this solution was used as sample solution 1. The inclusion was weighted appropriately (equivalent to about 10 μl volatile oil), and treated similarly, thus yielding the sample solution 2. The inclusion was weighted appropriately (equivalent to about 0.6 ml volatile oil), and the volatile oil was collected by reflux extraction. Afterward, 10 μl of the collected volatile oil were pipetted and treated similarly to the sample solution 1, thus yielding the sample solution 3. The volatile oil was mixed with β-CD physics (0.01 ml: 0.08 g) and then treated similarly to sample solution 1, thus yielding the sample solution 4. Then, 0.08 g of the inclusion was weighted appropriately, and treated similarly to the sample solution 1, thus yielding the sample solution 5. Afterward, 10 μl of the above five kinds of the sample solutions were pipetted, and then dropped in a silica G thin layer plate, with petroleum ether (60-90°C): Ethyl acetate (whilst. 5) as developing agents. The samples were removed and dried. Then, 5% of sulfuric acid vanillin solution was sprayed on the sample, and hot air was blown to the spot until color was clear, as shown in [Figure 1].
Figure 1: The thin layer chromatography spectrogram of volatile oil before wrapping (1), inclusion complex (2), inclusion complex of the volatile oil (3), volatile oil and the beta-cyclodextrin physical mixture (4) and beta-cyclodextrin (5)

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Ultraviolet spectrophotometry

The sample liquid was prepared by “3.7.1” and ultraviolet (UV) scanning was conducted within 200–400 nm, as shown in [Figure 2].
Figure 2: The ultraviolet spectrogram of volatile oil before wrapping (1), inclusion complex (2), inclusion complex of the volatile oil (3), volatile oil and beta-cyclodextrin physical mixture, (4) and beta-cyclodextrin (5)

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Differential thermal analysis

Thermogravimetric/differential Thermothermal Analysis was carried out on three samples of β-CD, β-CD, and lavender, and fennel volatile oil physical mixture and the inclusion complex. The scanning conditions were as follows: one aluminum metal sample plate was used as reference, and the samples were placed into the other aluminum metal sample plate. The heating speed was 10°C/min. The nitrogen flow speed was 20 mL/min. The scanning range was 30°C–400°C and the sample weight was approximately 5 mg. The thermogravimetry and differential thermal analysis (TG and DTA) heating curve of the three samples were recorded, respectively, as shown in [Figure 3] and [Figure 4].
Figure 3: The differential thermal analysis map of volatile oil (lavender, fennel mixture, and volatile oil) and beta-cyclodextrin physical mixture (1), beta-cyclodextrin (2) and inclusion complex (3)

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Figure 4: The thermal gravimetric analyzer map of volatile oil (lavender- and fennel-mixed volatile oil) and beta-cyclodextrin physical mixture (1), beta-cyclodextrin (2) and inclusion complex (3)

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Microscopic imaging method

As shown in [Figure 5], we took small samples of β-CD, β-CD, and lavender and fennel volatile oil physical mixture and the inclusion complex. Each of the above three samples were filled into a little water filling piece, respectively, placing them on a glass slide and observing the image with a microscope and photographic capture.
Figure 5: The microimaging of beta-cyclodextrin (a), volatile oil (lavender- and fennel-mixed volatile oil) and beta-cyclodextrin physical mixture (b) and inclusion complex (c) (10 × 10)

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  Results and Analysis Top


Determination result of the relative density of volatile oil

The relative densities measured by the three parallel tests were 0.944, 0.944, and 0.943, respectively, with an average value of 0.944.

Determination result of the blank recovery rate of volatile oil

The blank recovery rates of the three parallel experiments were 96%, 95%, and 94%, respectively, with an average value of 95%.

Response surface experiment results and analysis

Y1(inclusion rate of volatile oil) and Y2(yield of inclusion complex) were considered as evaluation indexes according to the comprehensive evaluation method to calculate the comprehensive score values Y. With comprehensive score values as effect value, the multiple linear regression equation of the comprehensive score value Y was calculated as Y = 0.9977Y1 + 0.374Y2. The experimental measured values were used in multiple linear regression equations of the experiment and the comprehensive score values were calculated. The results are shown in [Table 3].
Table 3: Box-Behnken design experiment result

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According to the regression analysis of the experimental data in [Table 3] by Design Expert 8.0.6.1 software, the equation of the quadratic multivariate regression model is: Y = 99.27 + 5.45A − 0.74 B + 0.61C − 0.098AB − 0.52AC + 0.20BC − 3.07A2 − 6.28B2 − 4.68C2

[Table 4] shows that the model is significant, and the model of comprehensive score is better than the actual experiment. In addition, the relationship between response quantity and independent variables is significant. The variance analysis in the table shows that the significant order of the response values is A > B > C, the interactive items (AB, AC, and BC) have no significant effect on the indicators, the quadratic terms (A2, B2, and C2) have extremely significant influence on the indicators, and the model also reached an extremely significant level, indicating that these three factors are not simple linear relationships to the comprehensive score of the study index.
Table 4: Box-Behnken design regression model analysis of variance-Y

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The experimental results were systematically analyzed, and the optimum inclusion process parameters of the inclusion complex of the β-CD inclusion of lavender and fennel volatile oil were obtained as follow: the ratio of volatile oil to β-CD was 8.13: 1, the inclusion temperature was 44.00°C, and the inclusion time was 1.03 h. It is predicted that the inclusion rate of the volatile oil obtained in this process was 69.71%, the recovery rate of the inclusion complex was 79.41%, and the comprehensive score was 99.27. According to the actual production conditions, the process parameters were corrected. The final inclusion conditions of β-CD were as follows: The ratio of volatile oil to β-CD was 8.13: 1, the inclusion temperature was 44.00°C, and the inclusion time was 1.0 h.

The response surface analysis of the different factors, the other two factors on the score of the comprehensive impact when A, B, or C are zero level, as shown in [Figure 6], and the three factors on the interaction of the experiment are shown in [Figure 7].
Figure 6: Box-Behnken response surface for the effect of inclusion temperature and β-CD: volatile oil on the comprehensive (a), inclusion time and β-CD: volatile oil on the comprehensive (b), inclusion time and inclusion temperature on the comprehensive (c)

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Figure 7: Contour map of the interaction between the inclusion temperature and β-CD: volatile oil (a), the interaction between the inclusion time and β-CD: volatile oil (b), the interaction between the inclusion temperature and inclusion time (c)

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As can be seen from [Figure 6]a, when the temperature is a certain value, the comprehensive score value increases rapidly with the increase of β-CD: volatile oil is formed and then slowly decreases. As can be seen from [Figure 6]b, when β-CD: Volatile oil presence is a certain value, the comprehensive score value increases with the increase of the inclusion time and then decreases. The overall trend is very smooth, indicating that the impact of the inclusion time on the experimental results is small. As shown in [Figure 6]c, when the inclusion time is short, the comprehensive score value increases slowly with the increase of the inclusion temperature at the beginning. When the inclusion time is longer, the comprehensive score value decreases with the increase of the inclusion temperature.

In [Figure 7]c, the contours of the contour lines indicating that the interaction effect between inclusion temperature and inclusion time is weak, have influence on the experimental results, but not significant enough. The contours of the contour lines in [Figure 7]a and [Figure 7]b are elliptic, indicating the interaction between β-CD: volatile oil presence and the inclusion temperature, and the interaction between β-CD: volatile oil presence and the inclusion time have significant effects on the experiment.

Verification experiment

In accordance with the above optimal parameters of lavender and fennel-mixed volatile oil was carried out in three parallel groups of the inclusion verification test. The results are shown in [Table 2]. As can be seen from [Table 2], the inclusion ratio of the volatile oil in the finished product obtained by the actual inclusion test differs from the predicted value by 1.13%, the inclusion complex recovery rate differs from the predicted value by 1.14%, and the comprehensive score value differs from the predicted value by 1.57%. The results show that the deviation of the predicted value from the actual value is small. The inclusion process parameters based on the BBD response surface method are accurate and reliable and have practical application value.

Characterization

Thin-layer chromatography analysis

It can be seen from [Figure 1] that the test liquid 1, the test liquid 3, and the test liquid 4 show spots of the same color at the corresponding positions, the test liquid 2 and the test liquid 5 are in the corresponding positions without any spots. This indicates that the inclusion complex has been formed; hence, the surface has no volatile oil residue, and there is no significant change in the main components before and after the inclusion of lavender and fennel volatile oil.

Ultraviolet Spectrogram analysis

As can be seen from [Figure 2], there is no characteristic absorption peak of volatile oil in the test liquid 2 and 5, while the test liquid 1, 3, and 4 have similar absorption peaks of mixed volatile oil in the same position. It shows that the volatile oil has been incorporated into the cavity structure of β-CD and has formed a new phase, and the physicochemical properties of the volatile oil have not changed under the inclusion action of an external force.

Differential thermal analysis

β-CD, volatile oil (lavender- and fennel-mixed volatile oil) and β-CD physical mixture and the inclusion complex differential thermal analysis are shown in [Figure 3]. As can be seen from the figure, β-CD has two characteristic peaks at 85.9°C and 316.9°C, which are the dehydration peak and the melting decomposition peak of β-CD. The physical mixture is a simple superposition between the volatile oil (Lavender- and fennel-mixed volatile oil) and β-CD. It also has two characteristic peaks at 79.8°C and 316.2°C, which were almost the same as the characteristic peaks of β-CD. The characteristic peaks of the inclusion complex were obviously different from those of the β-CD and the physical mixture. On the one hand, the dehydration peak was delayed to 95°C, indicating a new phase may be generated. On the other hand, the decomposition peak above 300°C was also different from the β-CD, the physical mixture, and it may also be related to the formation of a new phase. Moreover, the temperature span between the beginning and the end of the inclusion complex characteristic peaks increases, and the thermal entropy value decreased significantly. The inclusion complex showed a new thermodynamic characteristics in the entire DTA temperature scan range of the entire DTA.[12] Presumably, the lavender- and fennel-mixed volatile oil and the β-CD are not undergoing simple adsorption, but generated a new phase by the interactions between molecules.

Thermogravimetry analysis

[Figure 4] shows the thermal gravimetric analyzer map about β-CD, volatile oil (Lavender- and fennel-mixed volatile oil) and β-CD physical mixture, and inclusion complex. β-CD have two apparent weightlessness at 54.3°C and 304.4°C. Lavender- and fennel-mixed volatile oil and β-CD physical mixture also have two obvious weightlessne ss at 57.2°C and 306.7°C, which is approximately similar to β-CD. The inclusion complex had only one weightlessness at 289.9°C, before this temperature, the inclusion complex is very stable, indicating that a new phase had formed before and after the inclusion.[13],[14],[15],[16],[17]

Microscopic imaging analysis

The β-CD was an irregular, translucent cubic plate-like crystal, its particle size was relatively large and clearly defined as shown in [Figure 5]a. The lavender- and fennel mixed volatile oil and the β-CD physical mixture were black opaque oily substances as shown in [Figure 5]b. However, the oily substances were not seen in the inclusion complex of small particles containing black opaque as shown in [Figure 5]c. The results show that β-CD did not change only under the action of the external force; however, the morphology and color of β-CD inclusion lavender- and fennel-mixed volatile oil were all changed, indicating that a new phase was formed.


  Conclusion and Discussion Top


Conclusion

The optimal inclusion process was obtained using the response surface method: The ratio of β-CD and volatile oil was 8.13:1. The inclusion temperature was 44.00°C. The inclusion time was 1.03 h. By repeating the tests, three times according to the best process described above, the inclusion rate of the volatile oil was measured. The yield of inclusion complex and the comprehensive score value had little deviation from the predicted value. This shows that the process optimization method using Box–Behnken response surface method is simple and convenient, providing a reasonable and reliable fitting result. In this experiment, thin-layer chromatography (TLC), UV spectrophotometry, thermogravimetric/differential thermothermal analysis and microimaging were used to identify the inclusion complex of lavender and fennel. The result showed that the inclusion complex has been formed and the effect is better.

Discussion

Box–Behnken design was applied in this experiment. The response surface optimization method was applied to optimize the mixed volatile oil of lavender and fennel inclusion process. The results show that the design method is a good application in optimization of the volatile oil inclusion process. The experiment scheme is feasible, the rate of volatile oil inclusion and the yield of inclusion are high.

As shown by the results, when the dosage of β-CD is increased to a certain extent, the increase of the dosage (8:1) yield does not improve at all, similar to the inclusion time. The inclusion of high temperature is not good for the inclusion of volatile oil. This is associated with the high temperature, the time spent by the inclusion and the loss of volatile oil volatilization.

The traditional method of adding volatile oil is to be directly sprayed on the solid preparation (such as granules and tablets) on the surface. The method is very strict regarding the packaging material of the preparation. Considered the stability of the volatile components, volatile oil is easy to volatilize in the process of placement, so this prescription adopts the use the β-CD saturated water solution method to the inclusion of volatile oil, improving the volatile oil stability in the preparation, so as to ensure the quality.

The volatile oil was encapsulated in the β-CD molecular cavity to form a molecular capsule. The oxidation decomposition reaction can be prevented by avoiding the contact between the effective components of the volatile oil and the external environment, thus turning the powder into a liquid drug reducing the loss of volatile oil during formulation and storage, and improving the utilization of volatile oil and the stability of the preparation. In addition, the β-CD is an inexpensive pharmaceutical adjuvant, the inclusion of lavender- and fennel-mixed volatile oil can provide an experimental basis for the development of an abnormal black biliary maturation agent into a modern Chinese medicinal preparation.

β-CD is an inexpensive pharmaceutical adjuvant, the inclusion of lavender- and fennel-mixed volatile oil can provide an experimental basis for the development of an abnormal black biliary maturation agent.

Acknowledgments

My deepest gratitude goes first and foremost to Professor Dong-Xun Li, my mentor, for his constant encouragement and guidance. He has walked me through all the stages of the writing of this thesis. Without his consistent and illuminating instruction, this thesis could not have reached its present form.

Second, I would like to express my heartfelt gratitude to Professor Guo-Song Zhang, who have instructed and helped me a lot in experimenting for the past three years.

Last my thanks would go to my beloved family for their loving considerations and great confidence in me all through these years. I also owe my sincere gratitude to my friends and my fellow classmates who gave me their help and time in listening to me and helping me work out my problems during the difficult course of the thesis.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (no: 81660667).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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