**5.3 Present information consistently and "straight to the point"**

In the process of encouraging individuals to follow the proposed preventive actions [58–60], information should be presented in a straight-forward manner [16] and in "one voice". Moreover, messages should use simple language [61], and be consistent in terms of message content, as inconsistency can lead to confusion and undesired health outcomes: "A well-crafted national message [has] the potential to build unity around the goal of defeating the virus through behavior change, preferably with clear, unambiguous recommendations of what actions to take" ([61], 1736). *For example, when the crisis first surfaced, the Austrian government stressed the importance of staying home; after the first lockdown, when social distancing was in order, the campaign commonly referenced the baby elephant as a metaphor to remind individuals to keep their distance (of 1.5 m; see Figure 2).*


**5. Difference in and between Carbon and Silicate Oxide Coatings**

The current commercial carbon thin films have a slight, brownish to golden, tint [26]. Although this may restrict applicable product categories of carbon coated PET bottles, from the viewpoint of the visual quality of products, the degree of the tint appears to position within the scope of consumer acceptance, based on the commercial products of white wine (Figure 4) and edible oil categories in the Japanese market.

**Figure 4.** Example of DLC coated bottles for wine.

In case of beverage and liquor market in Japan, carbon coating is more often seen than silicate oxide coating in spite of the abovementioned disadvantage to carbon coating. The reason might be found in that carbon thin films are readily applicable to various product categories because carbon coating is inert to food and beverage solutions as long as the PET substrate is stable. On the other hand, some more remarkable limit in applicable product properties is known in typical silicate oxide coating. The gas barrier property of silicate oxide coatings may be decreased in contact with some solutions, for example, beverages of pH close to neutral [27].

C2H2 (acetylene) is the main material gas for carbon thin films for gas barrier-enhanced PET bottles. Derived from the hydrogen contained in acetylene molecules, the resultant carbon thin films contain hydrogen components up to 40% in atomic percentage. ERDA (Elastic Recoil Detection Analysis) analyses showed oxygen components up to 10% may be present in the carbon thin films [4,28], which is considered to be mainly derived from water vapor from PET substrates. The advantage of the use of acetylene lies in high deposition rates and economics, while CH4 (methane) is used in many studies [4,26]. At least carbon thin films derived from acetylene contain the carbon bonding of *sp*3, *sp*2, and *sp*1, based on XPS and FTIR studies [22]. In the Japanese market, carbon-coated PET bottles are derived from Kirin's DLC and Sidel's Actis™ technologies [14].

HMDSO (hexa-metyl-di-siloxan) and HMDSN (hexa-metyl-di-silazane) are the main material gases for silicate oxide thin films with aid of the controlled supply of oxygen. Based on the ratio in the mass flow of the material gas to oxygen and other conditions, the resultant thin films have different components consisting of silicate, carbon, oxygen, and hydrogen [29]. The components have impacts on gas barrier properties and stability in contact with beverage solutions, and sometimes also on tint. In the case with commercial gas barrier silicate oxide, thin films are totally colorless in visual observation. In the Japanese market, silicate oxide-coated PET bottles can be mainly seen in domestic edible oil and wine products, and rarely seen in imported carbonated water. Those bottles are derived from Toyo Seikan's Sibird™, Toppan's GL-C™, and KHS's Plasmax™ technologies [14].

From the viewpoint of the frequency of power used to cause the plasma states of material gas supplied, radio frequency (13.56 MHz) and microwave (2.45 MHz) are used in commercial technologies. The use of radio frequencies usually leads to a bi-electrode system, in other words, a type of capacitively-coupled plasma system, where sheath voltage and the resultant ion impact over the surface of the substrate can be controlled relatively precise manner [28]. It can be expressed that the use of these systems involves both merits and demerits to machine users. Examples of the merits are possible improvement in the performance of coating and stable application to relatively small or large containers, while those of the demerits are the possible increase of the change of mechanical parts for the application to containers of different shapes and sizes.

#### **6. Recent Advancement in Commercialized Technologies for Coating Plastic Containers**

In spite of the different nature of carbon and silicate oxide thin films as described above, it can be said that the difference between the two thin films is decreasing in the recent technical advancement.

It is obviously conceivable that the optimization of process conditions in parallel to the improvement in machinery has been performed in each technology, and as a result, deposition time has been shortened while the barrier properties of PET bottles coated are maintained or even improved. It is supposed that typical process conditions, including vacuum pressure, gas flow rate, and power application have been optimized. As a result, carbon coating has been less colored, and widened its applications (as shown in Figure 2). In the same way, silicate oxide coating has clarified and mitigated its limitation in applications, and widened its applications. In the case of the Japanese market, the use of coating technologies has been rapidly increased in recent years and, at present coating is the most abundant among technologies, compared to other gas barrier enhancement technologies applied to PET bottles [14].

An example of technological advancement has been found in the appropriate use of dielectric materials along electrodes in Kirin's DLC coating technology [30], and the modification of power frequency. Conventionally, this technology employed 13.56 MHz for power frequency and outer electrodes made of metal (conductive materials) parts only. Recently, power frequency was confirmed as one of the significant process parameters [28]. The use of 6 MHz for power frequency and outer electrodes fully or partially covered with dielectric material parts has been proposed in order to facilitate finding the appropriate process conditions for high gas barrier coatings (as shown in Figure 5) in addition to a decreased change of electrode parts for bottles of different shape and size. The results of the observation of coating thickness and plasma light emission indicate that the reason why 6 MHz power frequency showed the lowest gas barrier performance lies in the optimized spatial distribution of plasma. Compared to plasma produced with 13.56 MHz, where the plasma tends to concentrate around the neck part of the bottle, it seems that plasma with a lower frequency provides higher ion impacts to the PET substrate and the resultant secondary electrons modify the spatial distribution of the plasma to the direction of the bottom part of the bottle.

**Figure 5.** Example of the impact on power frequency to the performance of coated bottles. 500 mL PET bottles were coated with DLC using different power frequency ranging from 2.5 MHz to 13.56 MHz, and the oxygen transmission rate (OTR) of these bottles were measured. Optimized power frequency was found at 6.0 MHz in terms of OTR. The measurement of OTR was performed based on ASTM F1307 [31] method using an Oxran 2/21 device, Mocon Co., Ltd., (Brooklyn Park, MN, USA) under conditions of 23 ◦C and 90% relative humidity.

Another example has been found in the modification in the manner of material gas and oxygen supply during the coating process of KHS's Plasmax™. This technology is called Plasmax Plus™. Due to an extra carbon-rich layer formed on the conventional silicate oxide layer, the resultant coating can be stable in contact with solutions of pH close to neutral, which deteriorates the performance of coating based on the conventional process. Interestingly, the new coating manner requires no machinery modification [27].

#### **7. Possible Near Future Advancement in This Field**

The above description in this review mainly covered a brief history of gas barrier enhancement of PET bottles through plasma-assisted CVD techniques. On the other hand, a lot of effort has been made to other types of plastic containers and novel approaches to gas barrier enhancement.

Although the current era where PET bottles are the most abundant package format of rigid plastic containers is likely to last in this and the following decades because of their industrially-favorable balance between performance and economics, other plastic materials also have demands for functional thin film coating. Some polyolefins, such as PE (poly(ethylene)) and PP (poly(propylene)), are quite useful materials while the lack of oxygen and other barrier properties limits their benefits. For possible example, coated PP bottles or jars could keep the flavor quality of filled contents for certain extended periods of time in addition to high heat resistance, compared to PET containers, which are limited in applications below the boiling temperature of water.

The authors found a remarkable difference in the degree of oxygen gas barrier enhancement with DLC coating formed on various kinds of plastic film substrates, as shown in Table 5 and Figure 6. However, a positron annihilation [32] study by the authors indicates that, on these substrates, DLC coating can be formed homogeneously in terms of free volume, as shown in Figure 7. The positron annihilation method is based on a phenomenon where positrons implanted into a condensed matter annihilate with an electron and emits two 511-keV γ quanta. The spectra of γ energy, including the Doppler shift, are characterized by the S parameter, which mainly reflects changes due to the annihilation of positron-electron pairs with a low-momentum distribution. For amorphous materials, positronium (Ps: a hydrogen-like bound state between a positron and an electron) may form in open spaces (or free volumes). Figure 7 clearly shows that DLC films has a small S parameter, compared to polymer substrates, and that thin films of small free volume can function as barriers against gas

permeation. Empirically, packages made of PE and PP tends to have relatively rough surface, and rough surface is considered to lead to significant defects in coating. When the surface of them and PET bottles is observed using an atomic force microscopy, the *R*a of 1 μm square is usually 30–100 nm and less than 1 nm, respectively. A result of wet coating approach [33] supports this concept, where a specific type of organosilane materials placed between DLC thin films and PP substrates remarkably enhanced oxygen barrier property, even though the organosilane layer itself did not have a significant barrier property to the PP substrate. It should be noted that, when the surface of the organosilane layer is observed with an atomic force microscopy, the *R*a of 1 μm square is usually around 1 nm. In this case, the smoothed surface with an increased anti-crack property due to the organosilane layer caused the enhancement of the coating. These results suggest the interface between thin films and substrates plays a crucial role on the enhancement of gas barrier property with dense thin film coating, and technologies for surface conditions are considered to be a key for the future commercialization of coated containers made of various plastics such as PE and PP.

In the other way, a novel approach to gas barrier thin film coating has been proposed, where a hot wire or catalytic CVD technique is applied to bottle coating in an attempt to achieve decreased installing expenditure based on the simple configuration of coating machines compared to that of conventional plasma assisted CVD machines. Furthermore, the application of this technique can produce unique gas barrier coating to PET bottles, like an intermediate between carbon and silicate oxide thin films [34]. Since the machine installation cost seems to be the bottleneck to further distribution of thin film coating technologies, significantly low cost machinery may be a remarkable breakthrough in this field.

**Table 5.** List of plastic materials used for comparing the degree of gas barrier enhancement with DLC coating.

**Figure 6.** Comparison of (**a**) the oxygen transmission rate (OTR); and (**b**) BIF of DLC coating on different plastic films. Samples of 40 mm square size were placed on the center of body part of 500 mL PET bottles were coated and measured based on the ASTM D3985 method using Oxtran 2/21 devices, Mocon Co., Ltd. (Brooklyn Park, MN, USA), under conditions of 23 ◦C and 90% relative humidity, in the same manner in a previous study [28].

**Figure 7.** Depth profile of the positron annihilation of DLC coated samples. The *S* parameter of DLC coating (see the region of less than 1.5 keV) and plastic substrates (see the region of more than in 5.0 keV) was measured in the same manner in a previous study [28] for observing the relative free volume of DLC coating layers.

The above discussion on near future technologies indicates a high potential of further advancement in thin film coating technologies for hollow plastic containers in this field, including applications to food and beverage industry.

**Acknowledgments:** We would like to express our gratitude to Tetsuya Suzuki, and Taku Aoki, and Kazuhisa Tsuji, Keio University, Japan, for profound discussion. We are also thankful to all the staff related to this article in University of Tsukuba, Japan, and Kirin Co. Ltd., Tokyo, Japan.

**Author Contributions:** Masaki Nakaya conceived and designed the experiments on DLC coating to PET bottles in Figures 5 and 6, Table 5, and a part of Table 4. Masaki Nakaya and Akira Uedono conceived and designed the experiments in Figure 7. Masaki Nakaya and Atushi Hotta conceived and designed the experiments using organosilane. Masaki Nakaya performed the experiments in Figures 5 and 6, and a part of Table 4. Masaki Nakaya and Akira Uedono performed the experiments in Figure 7. Akira Uedono analyzed the data in Figure 7. Atsushi Hotta contributed PP substrate materials and analysis tools in the experiments using organosilane. Masaki Nakaya wrote the paper.

**Conflicts of Interest:** The authors declare no conflict of interest.


**Abstract**

Our ability to investigate both the intrusive and extrusive parts of individual volcanoes has evolved with the increasing quality of seismic reflection datasets. Today, new seismic data and methods of seismic interpretation offer a unique opportunity to observe the entire architecture and stratigraphy of volcanic systems, with resolution down to tens of meters. This chapter summarises the methods used to extract the geomorphic aspects and spatio-temporal organisation of volcanic systems buried in sedimentary basins, with emphasis on the utility of 3D seismic reflection volumes. Based on descriptions and interpretations from key localities worldwide, we propose classification of buried volcanoes into three main geomorphic categories: (1) clusters of small-volume (<1 km3 ) craters and cones, (2) large (>5 km3 ) composite, shield and caldera volcanoes, and (3) voluminous lava fields (>10,000 km3 ). Our classification primarily describes the morphology, size and distribution of eruptive centres of buried volcanoes, and is independent of parameters such as the magma composition, tectonic setting, or eruption environment. The close correlation between the morphology of buried and modern volcanoes provides the basis for constructing realistic models for the facies distribution of igneous systems buried in sedimentary strata, establishing the principles for a new discipline of seismic-reflection volcanology.

**Keywords:** seismic volcanostratigraphy, seismic geomorphology, buried volcanoes, volcanic landforms, igneous plumbing systems, seismic-reflection volcanology


**Figure 8.**

*Atomic force microscopy image shows a panoramic view of the interdendritic region of the irradiated zone where the distribution and size of the induced nanoparticles can be observed as a result of the interaction process of the irradiation with the protrusions observed in* **Figure 2***.*


Anthropometric Characteristics

Height was measured without shoes with the use of a stadiometer, and body mass was measured with a calibrated digital scale (Seca 208 and Seca 710, Hamburg, Germany). Body mass index was calculated as the ratio of body weight to the squared standing height (kg/m2). The length of each lower extremity was measured as the distance between trochanter major to the floor with the participants in standing position. The distance between tibiofemoral joint cleft and medial malleolus was determined as calf length.

#### *2.3. Gastrocnemius Medialis Architecture and Ankle Joint Angle at Rest*

In order to avoid trigonometric estimations or multiple scans along the muscle length to be assembled [27], in the present study, panoramic ultrasound images were obtained, via extended-field-of view imaging, along the fascicle length of GM.

All ultrasound measurements were performed in the morning and after athletes remained in a prone position on the examination bed, with their ankles hanging loosely on the outside of the bed for at least 20 min [28]. Muscle architecture of the right leg GM, (dominant leg that is in stance while kicking a ball) was obtained with a 10 MHz linear probe (38 mm) via extended field of view mode (Product model Z5, Shenzhen, Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China). Ultrasound images were recorded at the medial and the distal part of the GM muscle belly: one-third and half of the distance from the popliteal crease to the center of the medial malleolus, respectively. These points were marked on the skin using an echo-absorptive tape that served as reference marker [28] (Figure 1). In order to measure musculotendinous junction (MTJ) displacement, MTJ was located by real-time static ultrasound imaging and marked on the skin by an echo-absorptive tape, as well. The transducer was orientated perpendicular to the skin and parallel to the fascicles to minimize perspective and parallax measurement errors [29]. A probe path (dashed line) was drawn on the skin with a permanent pen by using static ultrasound according to the fascicle path seen from the ultrasound image. A single view was taken by moving continuously the probe in a slow and steady rate along the marked path. For each part of the muscle (medial and distal), three different fascicle lengths were measured from the deep aponeurosis to the superficial aponeurosis with a linear trace. Where the muscle fascicles met the lower aponeurosis the respective pennation angles were measured. The average of the lengths and angles of the three fascicles was used for statistical analysis for each part of the GM. The distance between the superficial and deep aponeuroses was determined as muscle thickness. Two consecutive measurements for each part of the muscle were assessed, and the average value was used for further analysis. All images were analyzed with image analysis software (Motic Images Plus 2.0, Motic, Hong Kong, China). Test-retest reliability was determined by using the intraclass correlation coefficient on 6 participants, on two separate days. The ICC (two-way random effects) for muscle fascicle length was 0.93 (95% CI: 0.576–0.990, *p* = 0.000), for muscular thickness it was 0.90 (95% CI: 0.474–0.984, *p* = 0.001), and for pennation angle, 0.95 (95% CI: 0.689–0.993, *p* = 0.001).

**Figure 1.** Panoramic sonographic image of gastrocnemius medialis of a rhythmic gymnast (top panel) and a volleyball player (bottom panel) at rest and during stretching showing fascicle length and pennation angle at the mid-belly and at the distal part of GM. MTJ: muscle-tendon junction.

Ankle joint angle at rest was also measured with the athletes lying in a prone position, with their ankles hanging loosely o ff the bed. Resting ankle joint angle was defined as the angle created by the intersection of the femur-tibia to lateral malleolus line and lateral malleolus to fifth metatarsal line [19]. Reflective markers were placed on these anatomical points in order to define the ankle angle using a digital camera (Casio Exilim Pro EX-F1, Shibuya, Tokyo, Japan). Image analysis was performed via a free software (Tracker 4.91 © 2016 Douglas Brown). Intra-class correlation coe fficients for resting ankle angle was 0.98 (95% Confidence Intervals (CI): 0.833–0.998, *p* = 0.000).

#### *2.4. Gastrocnemius Medialis Architecture and Ankle Joint Angle during Ankle Dorsiflexion Stretching*

Panoramic ultrasound images from the two parts of the GM muscle belly (medial and distal) were obtained following the method described above. Reflective motion analysis markers, and echo-absorptive tapes remained on the skin, and the drawn path (dashed line) of the resting measurements was used, to re-assess the regions of interest. Following two minutes of standing, all athletes performed a slow, passive standing dorsiflexion stretching, for one minute. Five seconds before the end of the stretching intervention a pause was imposed to capture still images. To obtain GM ultrasound images during stretching, the probe was placed 38 mm above the skin marker that identified the middle part of the muscle belly. In addition, 'MTJ displacement' was defined as the di fference between the MTJ marker at rest and MTJ point during stretching (Figure 1).

Ankle dorsiflexion stretching while standing is commonly performed in sport practice [30], and the athletes were familiar with it. Stretching was performed with the athletes barefoot. Athletes were instructed to relax while they passively stretched their ankle plantar flexors, in a slow and continuous manner. The foot to be tested (right) was placed on the midline of a marked area on the floor, and the left foot was placed forward at step-length distance. The end point of standing dorsiflexion stretching was defined as the point that the athletes felt discomfort without lifting their heel and with no pelvic rotation. The athletes put their hands against the wall to maintain balance and were asked to keep the extended position of their hip and knee joints, during stretching [30]. Stretch intensity was indicated by the athletes using the 0–10 Wong-Baker FACES Pain Scale for children [31] to ensure that stretch achieved the point of discomfort (~8 on a scale of 0–10). During the execution of the stretch, participants were instructed to reach a pain of discomfort level of 8 in the scale of 0–10, and thus they held the stretch at exactly this perceived intensity. The psychometric properties of this commonly used pictorial scale assessing acute pain have been found to be appropriate for children over the age of 3 [31]. Six faces depict di fferent expressions, ranging from "no hurt" to "extremely upset from pain". A digital camera (Casio Exilim Pro EX-F1, Shibuya, Tokyo, Japan) was placed perpendicular to the plane of motion of the right leg in order to record the standing ankle dorsiflexion angle. Stretching ankle joint angle was analyzed using reflective markers placed on the knee, ankle and fifth metatarsal and calculated using free software (Tracker 4.91 © 2016 Douglas Brown). Maximal standing dorsiflexion was defined as the intersection of a line joining the knee and ankle markers and horizontal (a line crossing the heel and the fifth metatarsal).

#### *2.5. Statistical Procedures*

Descriptive statistics were calculated. Shapiro-Wilks test checked for normality of data distribution. Pearson correlations coe fficient (*r*) detected linear relations between the examined variables. Unpaired T-test examined di fferences between groups in anthropometry and architectural characteristics of GM at rest. A two-way ANOVA (time x group) with repeated measures for time (rest or stretch) and group (flexibility trained vs. not trained) was conducted separately for the medial and the distal part of the muscle, to examine the e ffect of stretching on fascicle lengths, pennation angles and thicknesses, and ankle joint angle. A Tukey post-hoc test was performed when a significant main e ffect or interaction was observed (*p* < 0.05). E ffect sizes calculation for pairwise comparisons was performed with Cohen's *d* [32]. To assess test-retest reliability the intra-class correlation coe fficients (ICCs) were used. Statistical

significance was set at *p* < 0.05. Statistical analyzes were conducted using SPSS (SPSS Statistics Version 25.0, IBM corporation, Armonk, NY, USA).


**4. Resistance on aminoglycosides**

Aminoglycoside drugs have been part of the fight against *E. coli* pathogenic species, however with the changes in the phenotype of the *E. coli* isolates for example the ever-evolving changes in the ESBL structure as well as the genes that the aminoglycoside drugs encode for susceptibility [26].

### **4.1 Aminoglycoside mode of action**

Aminoglycosides are bactericidal agents that inhibit the synthesis of bacterial proteins through the interruption of the ribosomes. They interfere by binding

to the 30S and 50S ribosomal subunits, they inhibit the translocation process of moving the peptidyl-tRNA from the A site to the P site thereby causing mRNA misreading in that way denouncing the translation process forwarding zero protein synthesis, which entails no budding, multiplication and denouncing perpetuation and survival [26].

#### **4.2 Mechanism of resistance on aminoglycoside drugs**

There are more than 50 types of aminoglycosides modifying enzymes which include, acetyltransferases (aac), phosphotransferases (aph) and nucleotidyltransferases (ant), these enzymes are capable of modifying aminoglycosides at their respective drug recognition sites [27]. The main cause of resistance to the aminoglycoside drugs is that more than one aminoglycoside modifying enzymes may be found in a single isolate bringing in a high probability rate of resistance to the drugs [28].

Mancin [28], conducted a population analysis of genetic and enzymatic resistance of *E. coli* to aminoglycosides, they concluded that the genes aac(6′) and aac(3) can cause significant resistance to amikacin and kanamycin. Another study on genes was conducted by Bodendoerfer [29], where they concluded that in Switzerland the most prevalent resistance genes included the aph(3′)-la, aac(3)-lld and aac(6′)-lb-cr, they also alluded that the genes tend to change in their structural arrangement which is a mediated by the action of the transposon mechanisms of the resistant genes. The mechanisms of the jumping gene continues to be a threat to human health, because the transposon genes can be transferred to the next cell through transformation and conjugation processes which will cause the development of more resistant genes prior to transcription and translation, this information has caused migraine headaches to researchers and scientists who are working tirelessly in the attempt of denouncing resistance [29].

### **5. Conclusion**

*E. coli* bacteria continues to be a nuisance in the medical field bringing endless prospects in resistance against the drugs that are used in an attempt of eradicating the bacteria. The bacterial species has so many different forms of isolates that differ from one another both structurally and genetically, hence the drug that is susceptible to one *E. coli* may be found ineffective to another *E. coli* variant species. The review showed that there are many different mechanisms in which *E. coli* strains are becoming more and more difficult to treat, some evolving to possess enzymes that work in a conjunctive manner to denounce the effect of the drug.

The evolution brings forth the production of many different isolates with different protein structures for drug resistance and allows perpetuation and survival. This alarms for continuous assessments to provide information on the drugs and the interference with the pathogenic microorganism, how the bacteria respond in susceptibility, which will act as a fortress in tracing the reason behind resistance tomorrow. Studies are of importance to help and provide practical proof on how to tackle pathogens which will aid in improving health, denouncing long hospital stays and even patients from succumbing to infections caused by the microorganism.

### **Conflict of interest**

The authors declare no conflict of interest.

*Mechanisms of Antimicrobial Resistance of* E. coli *DOI: http://dx.doi.org/10.5772/intechopen.101671*


**1. Introduction**

*Angelica gigas* Nakai (AGN) is one of the most important traditional herbal medicinal plants in Korea. AGN possesses abundant nutraceutical properties, with strong antioxidant capacity [1]. The roots of AGN contain coumarin derivatives decursin (D) and decursinol angelate (DA) [2] which have several pharmacological properties such as anti-amnestic activity [3] anti-bacterial action [4], anti-allergic effect [5], and anti-tumor activity [6]. AGN is mainly used for the treatment of menopausal syndromes in Korea; therefore, it is called 'female ginseng' [7].

Plant bioactive compounds have a strong intermolecular covalent bond with large molecular weight. This complex molecular structure makes them less functional [8]. Many methods, including mechanical, chemical, and radiation approaches, are being employed to improve the plant antioxidant profiles through the stretching and bending of molecules. Among those, far infrared irradiation (FIR) is an efficient and convenient method to liberate the molecules from their complex crystalline structure and reduce intermolecular energy [9]. FIR is a subdivision of the electromagnetic spectrum which is used in the food processing industry to induced biological activities and prevent food quality degradation [10].

FIR has the capacity to transfer heat through molecular vibration to the center of the materials without degrading the constituent of the molecules. FIR cleaves covalent bonds and liberates antioxidants such as phenolic acids, flavonoids, tannins, and carotenoids [7,10].

It is reported that FIR increases the quantities of nutraceutical compounds, while improving antioxidant property, anti-inflammatory, and inhibitory activity in the A549 Cell line in *Chrysanthemum indicum* L. [11]. In particular, FIR enhances phenolic content and antioxidant capacity of buckwheat flour [12], sprouting bean flour [13], citrus cack [14], and rice hull [15]. However, to the best of our knowledge, there are no reports published on the effect of FIR on nutraceutical compounds in AGN. Therefore, the objective of this study was to induce nutraceutical compounds and increase antioxidant capacity through the application of FIR treatment in AGN powder.

#### **2. Materials and Methods**

#### *2.1. Application of FIR Treatment and Preparation of AGN Extract*

*Angelica gigas* Nakai (AGN) was purchased from Pyeongchang local market, Korea. AGN was dried in an oven at 50 ◦C for 24 h and powdered using a grinder. The powder was meshed using a 200 μm sieve to obtain a uniform particle size of the powder. Two grams of powder were mixed with 4 mL of water in a glass petri dish and exposed to an FIR dryer (HKD-10; Korea Energy Technology, Seoul, Korea) at 120, 140, 160, 180, 200, 220 and 240 ◦C for 30 min. Afterward, 1 g of the treated powder and control sample (without FIR application) was suspended in 100 mL of 80% ethanol and kept over-night in a shaker at room temperature. The extracts were filtered through Advantech 5B filter paper (Tokyo Roshi Kaisha Ltd., Saitama, Japan) and dried using a vacuum rotatory evaporator (EYLA N-1000, Tokyo, Japan) in 40 ◦C water bath to obtain crude extracts. The crude extracts were freeze-dried to achieve a moisture content of <3–5%. Dried crude extracts were diluted using 80% ethanol to prepare 1000 mg/L stock solution and kept at −20 ◦C for further analysis.

#### *2.2. Estimation of Total Phenolic Content*

Total phenolic (TP) content of FIR treated and control AGN samples were determined by the Folin Ciocalteu assay [16]. In brief, a sample aliquot of 1 mL of extract (1 mg/mL) was added to a test tube containing 0.2 mL of phenol reagent (1 N). The volume was increased by adding 1.8 mL of deionized water and the solution was vortexed and left for 3 min for reaction. Furthermore, 0.4 mL of Na2CO3 (10% in water, v/v) was added and the final volume (4 mL) was adjusted by adding 0.6 mL of deionized water. The absorbance was measured at 725 nm after incubation for 1 h at room temperature. The TP content was calculated from a calibration curve using gallic acid and expressed as mg of gallic acid equivalent (GAE) per g dry weight (dw).

#### *2.3. Determination of Total Flavonoid Content*

The total flavonoid content (TF) content was quantified according to Ghimeray et al. [12] with slight modifications. Shortly, a 0.5 mL aliquot of the sample (1 mg/mL) was mixed with 0.1 mL of 10% aluminum nitrate and 0.1 mL of potassium acetate (1 M) solution. To this mixture, 3.3 mL of distilled water was added to make the total volume of 4 mL. The mixture was vortexed and incubated for 40 min. The TF content was measured using a spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan) at 415 nm. The TF content was expressed as mg/g coumarin equivalents on a dry weight basis.

#### *2.4. HPLC Analysis of AGN Extract with or without FIR Treatments*

An HPLC system (CBM-20A, Shimadzu Co, Ltd., Kyoto, Japan) with two gradient pump systems (LC-20AT, Shimadzu), a C18 column (Kinetex, 100 × 4.6 mm, 2.6 micron, Phenomenex), an auto-sample injector (SIL-20A, Shimadzu), a UV detector (SPD-10A, Shimadzu) and a column oven (35 ◦C, CTO-20A, Shimadzu) were used to analyze D and DA. Solvent A was water with 0.4% formic acid, and solvent B was acetonitrile. A gradient elution was used (0–15 min, 33–45% B; 15–30 min, 45–55% B; 30–40 min, 55–80% B; 40–45 min, 80–33% B). The flow rate was 1.0 mL/min, the injection volume was 10 μL, and the detection wavelength was 329 nm. The standard samples for the D and DA analysis were

prepared at concentrations of 10, 20, 40, 60 and 80 μg/mL. As D and DA are the main active ingredients of the AGN therefore, they were separated and then quantified by HPLC.

#### *2.5. Antioxidant Capacity Analysis*

#### 2.5.1. DPPH Free Radical Scavenging Capacity

The antioxidant capacity was determined on the basis of the scavenging activity of the stable 2, 2-diphenyl-1 picryl hydrazyl (DPPH) free radical according to methods described by Braca et al. [17]. The DPPH solution was prepared (5.914 mg of DPPH powder dissolved in 100 mL of methanol) to maintain an absorbance range of 1.1–1.3 by spectrophotometer. Briefly, 1 mL of stock solution was added to 3 mL of DPPH solution. The blank sample was prepared with 1 mL of distilled water instead of stock extract in 3 mL of DPPH solution. The mixture was shaken vigorously and left to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using a spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan). The percent inhibition activities of the treated and control AGN samples were calculated against a blank sample using the following equation:

Inhibition (%) = [(blank sample − extract sample)/blank sample] × 100.

#### 2.5.2. Ferric Reducing Antioxidant Power Assay (FRAP)

The reducing power of the samples was estimated according to the FRAP assay as described by Yu et al. [18]. In brief, 1 mL of stock solution (1 mg/mL) was mixed with 1 mL of 0.2 M phosphate buffer maintaining a pH of 6.6. The mixture was then incubated at 50 ◦C for 20 min. After incubation, 1 mL of trichloro-acetic acid (TCA) was added to the solution and centrifuged at 3000 rpm for 10 min. The collected supernatant was diluted with distilled water at 1:1 ratio. Finally, 0.25 mL of 0.1% ferric chloride was added and the absorbance was measured at 700 nm by a spectrophotometer.

#### 2.5.3. Phosphomolybdenum Method (PPMD)

The total antioxidant capacity of AGN stock solution (1 mg/mL) was assayed according to the PPMD method described by Prieto et al. [19]. In brief, 1 mL of stock solution was added with 3 mL of 0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate solution. The reaction mixture was incubated at 95 ◦C for 150 min. The absorbance of the mixture was measured at 695 nm by spectrophotometer against a blank. The antioxidant capacity was expressed as the absorbance of the sample.

## **3. Statistical Analysis**

All data were expressed as mean ± SD of triplicate measurements. The obtained results were compared among the different FIR temperature using a paired *t*-test in order to observe the significant differences at the level of 5%. The paired *t*-test between the mean values of the treated samples and control were analyzed by MINITAB (version 16.0).

#### **4. Results and Discussion**

#### *4.1. Effect of FIR Irradiation on Total Phenolic and Flavonoid Contents of AGN Powder*

Total phenolic and total flavonoid content of the FIR treated and control AGN was demonstrated in Table 1. It is shown that total phenolic (22.65 mg/g) and total flavonoid content (7.87 mg/g) was significantly increased at 220 ◦C of FIR treatment which is two times higher than of the control (12.75 mg/g, 2.51 mg/g, respectively). In the same way, it is shown in Table 2 and Figure 1 that the quantities of the main active ingredients decursin (62.48 mg/g) and decursinol angelate (41.51 mg/g) were about two and 1.5 times higher at 220 ◦C than the control (38.70 mg/g, 27.94 mg/g, respectively).

**Table 1.** Total phenolic (TP) and total flavonoid (TF) content of *Angelica gigas* Nakai treated by FIR irradiation.

Each value is expressed as the mean ± SD (*n* = 3). Values labeled with different letters in a column are significantly different (*p* < 0.05). GAE: gallic acid equivalent; CUE: coumarin equivalent; dw: dry weight.

**Table 2.** HPLC quantification of decursin and decursinol angelate content of *A. gigas* Nakai treated by FIR irradiation.

Each value is expressed as the mean ± SD (*n* = 3). Values labeled with different letters in a column are significantly different (*p* < 0.05).

It is stated that FIR is magnificently applied in the drying of various food materials [20]. FIR creates internal heating via molecular vibration of the materials i.e., molecules absorb the radiation of certain wavelengths and energy, causing excited vibration [21]. FIR significantly induced plant secondary plant metabolites through the breakdown of a covalent bond of long-chain polymers [22]. Previous studies observed that FIR application increased phenolic content in soybean sprout powder [13], rice hull [15], ginseng, garlic, tomato, grapes, and onion [22]. In our study, the highest phenolic and flavonoid compounds, including D and DA, were attained at 220 ◦C of FIR treatment. Since D and DA have several pharmacological properties, therefore, FIR would be a suitable stimuli to enhance these compounds.

Previously, Ghimeray et al. [12] obtained the highest quantities of total phenols, total flavonoids, and quercetin content of buckwheat at 120 ◦C for 60 min of the FIR treatment. On the other hand, Azad et al. [13] observed the highest phenolic and isoflavonoid content in soybean sprout powder at 120 ◦C with an exposure time of 120 min. In our study, the bioactive compounds content decreased as the FIR temperature was increased. The FIR treatment at 220 ◦C found to be more adventurous in this study. However, Adak et al. [23] showed that the content of phenolic compounds and anthocyanins in strawberries was reduced when the FIR temperature was above 80 ◦C. Therefore, it might be assumed that the optimum temperature and exposure time of the FIR depends on the plant materials.

**Figure 1.** HPLC chromatogram of decursin and decursinol angelate of control (above) and treated AGN at 220 ◦C (down).

#### *4.2. Effect of FIR Irradiation on Antioxidant Capacity of AGN Powder*

Plant bioactive compounds have an enormous antioxidant capacity due to their redox properties which allow them to act as reducing agents, hydrogen donators, metal chelators and single oxygen quenchers [24].

The effect of FIR on the free radical antioxidant capacity of the AGN powder was measured by the DPPH, FRAP and PPMD method. DPPH is a stable free radical compound widely used to test the free radical scavenging ability of various materials [25]. FRAP measures the reducing potential of an antioxidant reacting with a ferric tripyridyltriazine complex, producing a colored ferrous tripyridyltriazine. PPMD is based on the reduction of Mo (VI) to Mo (V) by the sample analyte and the subsequent formation of a green phosphate/Mo(V) complex [19].

It was shown that the antioxidant capacity of the AGN was increased by FIR treatment. Antioxidant capacity was found to be two times higher at 220 ◦C (79%) of FIR compared to control (37%), according to the DPPH analysis. In the FRAP method, antioxidants cause the reduction of the Fe3+/ferricyanide complex to the ferrous form and activity is measured as the increase in the

absorbance at 700 nm. In this assay, the yellow color of the test solution changes to various shades of green and blue depending on the reducing power of antioxidant samples [26]. In FRAP assay, the absorbance was three times higher at 220 ◦C of FIR treatment compared to control. On the other hand, the absorbance was four times higher in treated AGN at 220 ◦C compared to control measured by PPMD. The reducing power of the treated sample increased with increasing concentrations in a strongly linear manner. It is clearly shown that antioxidant properties of the AGN powder were increased at 220 ◦C FIR in all analytical methods i.e., DPPH, FRAP, and PPMD (Figures 2–4).

**Figure 2.** DPPH free radical scavenging activity of *A. gigas* Nakai treated by FIR irradiation. Each value is expressed as the mean ± SD (*n* = 3). Different lowercase letters within the row indicate significant differences (*p* <0.05) according to ANOVA. FIR: far infrared irradiation.

**Figure 3.** Reducing power of *A. gigas* Nakai treated by FIR irradiation (FRAP assay). Values are the mean ± SD (*n* = 3). Different lowercase letters within the row indicate significant differences (*p* <0.05) according to ANOVA. FIR: far infrared irradiation.

**Figure 4.** Total antioxidant capacity of *A. gigas* Nakai treated by FIR irradiation (PPMD assay). Values are the mean ± SD (*n* = 3). Different lowercase letters within the row indicate significant differences (*p* <0.05) according to ANOVA. FIR: far infrared irradiation.

Previous studies showed that FIR increased the antioxidant capacity of plant food materials [12,13,15]. It was also reported that total phenolic content and antioxidant capacity has a highly significant linear correlation [23]. The increases in antioxidant activity of treated samples is due to the increase of the total polyphenol and flavonoid compounds [27].

It is reported that D and DA are not directly correlated to the total antioxidant capacity in AGN; however, the increased content of D and DA improve the pharmacological profile [27]. Significant correlations between the content of total phenolic compounds and antioxidant capacity (R2 = 0.852) were observed in this study (Figure 5), and are supported by the findings of Cai et al. [28] and Djeridane et al. [29].

**Figure 5.** Linear regression between total phenolic content and antioxidant capacity (DPPH assay).


**1. Introduction**

Until recently, electrophysiological evaluations were performed exclusively with nonverbal stimuli such as clicks and tone bursts which allow rapid and synchronous stimulation of neurons. However, the use of verbal stimuli, such as speech sounds, allows a more accurate analysis of the auditory system, especially if the aim is to investigate how the system decodes speech sounds involved in daily communication. Verbal and nonverbal stimuli are decoded in different ways and follow different trajectories through the central auditory nervous system.

Human communication consists predominantly of verbal stimuli, and it is important to understand how verbal sounds are coded at various levels of the auditory system. The need to develop research methods that are objective and accurately represent daily listening led to the development early this century of electrophysiological tests for measuring how speech sounds are perceived [1, 2]. Subsequently, a number of research groups have focused their efforts on using complex stimuli such as speech for diagnostic purposes [3–10].

The initial studies were performed in animal models [11] aiming to evaluate how the temporal and spectral properties of verbal stimuli were coded, and later human responses were also analyzed [12]. Among the electrophysiological procedures for investigating the processing and coding of verbal sounds, we highlight the frequency following response (FFR).