**3. Assay of cooked rice elongation**

Ten selected grains were soaked in distilled water for 30 min which were then placed between two pieces of wet filter paper in a petri dish filled with an appropriate amount of water. The dish was then placed in a covered container and the grains were cooked by steaming over boiling water for 10 min and simmering for 10 min (with power off). The cooked rice grain was transferred onto a piece of dry filter paper at the bottom of a fresh petri dish, which was placed in a desiccator with a constant temperature (19°C). Then the CRL was measured [11].

Ten randomly chosen intact polished grains were boiled in a water bath for 7 min after soaking in 7.5 ml of distilled water for 20 min. The cooked rice grains were then placed on absorbent paper for 15 min, and the boiled grain length (BGL, mm) and width (BGW, mm) were measured using the same scanner. The treatments were repeated twice, and the average grain length and width before and after cooking were calculated for estimation of elongation index.

The length of 10 whole rice kernels after cooking was measured by using the micro-scale, and the average kernel length determined. Kernel elongation ratio was calculated by dividing the average length of cooked kernel by the average length of the raw (uncooked) rice.

An inter-laboratory collaborative test was conducted for measurement of grain elongation of milled rice during cooking in 19 laboratories in 16 countries. Details of individual laboratory methods for measurement of grain elongation given below [9].


*Article* **Interactive Web-Map of the European Freeway Junction A1**/**A4 Development with the Use of Archival Cartographic Sources**

## **Dariusz Lorek \* and Tymoteusz Horbi ´nski**

Department of Cartography and Geomatics, Faculty of Geographical and Geological Sciences,

Adam Mickiewicz University, 61-712 Pozna ´n, Poland<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>Tel.: +48-61-829-6249

Received: 24 June 2020; Accepted: 9 July 2020; Published: 14 July 2020

**Abstract:** In the article, authors have analyzed cartographic materials presenting the spatial development of Gliwice with the use of multimedia tools. The materials prove that this area has played an important part in the road system of the region, country and even part of Europe since the 19th century. The six maps from the studied area were analyzed e.g., the Urmesstischblätter map, polish topographic maps, and the OpenStreetMap. Based on these maps and their legends, vectorization of the main roads of the analyzed area was carried out. The evolution of the main road corridors on the six maps was analyzed with respect to the location of the European freeway junction (A1/A4), constituting a basis for the web map. According to the authors, the use of the interactive web map is the most comprehensive method of all technologies used by modern cartography. Spatial data collected from different cartographic publications (from the first half of the 19th century till the present) consider the most significant aspects of changes in the road network of the analyzed area in a detailed and user-friendly way.

**Keywords:** freeway junction; topographic maps from the 19th century; interactive web map; Leaflet; GeoJSON

## **1. Introduction**

Topographic maps include the general geographic information, which determines their huge informational potential as far as the comprehensive study of changes in the environment and spatial structures is concerned [1]. The symbols standing for major roads were included in the legends of such maps, the editions from both the 19th and 20th centuries, at the very beginning. Major roads constitute a constructional basis for the diversely used land, marking its structure. Moreover, the road system, with its density or differences in quality, reflect the stage of development of a given area. Successive map editions demonstrate the evolution of road networks and typical changes in land use forms. The information included in historic maps constitutes a valuable source of information helpful in reconstructing historic states of space both in terms of individual objects and phenomena and entire areas defined as integrated settlement structures, thoroughfares, and land use forms [2].

The analysis of cartographic materials presenting the spatial development of Gliwice proves that this area has played an important part in the road system of the region, country and even part of Europe since 19th century [3]. Obtaining and visualizing spatial data concerning major roads and how they crossed over a span of nearly two hundred years is significant in order to be able to demonstrate changes in space use. The roads, mainly the 'Gliwice So´snica' freeway junction connecting international east–west and north–south corridors, significantly increased the importance of the area (A1 and A4 highway).

Modern cartographic forms, such as multimedia cartography, offer a wide range of opportunities to present and compile spatial data, i.e., AR (Augmented Reality) and VR (Virtual Reality) [4–6], data from the low flight level [7] or interactive web maps [8–10]. The aim of the research described in this article was to verify the hypothesis of whether or not it is possible to demonstrate changes occurring in space and to reconstruct historic states of space, using modern multimedia technologies and cartographic sources that present the network of the most relevant roads of the region from the mid-19th century to the present day.

## **2. Methodology**

#### *2.1. Study Area*

time, was used.

improved surveying instruments).

2.2.1. Urmesstischblätter Map

The study concerns the county of Gliwice located in southern Poland (Figure 1) which has constituted an important industrial center based on coal and ore mining and processing for over 150 years. Spatial development of the region was strictly linked with the processes of industrialization and urbanization that were constantly progressing since the mid-19th century, manifesting themselves mainly through buildings (houses, housing estates, mines, plants and factories) and road networks (roads, railroads, river/inland transport). The fact that the old city was located upon a river that played a significant part in transport was an important advantage of the city, increasing its role in the region [11,12]. Currently, despite economic transformations and the decrease in the role of the industry in the late '80s, the region has retained its character and form of development shaped by previous decades. At the same time, one can observe some changes in land use resulting from current urbanization processes. *ISPRS Int. J. Geo-Inf.* **2020**, *9*, x FOR PEER REVIEW 3 of 14

**Figure 1.** Freeway junction A1/A4 in Gliwice (Poland). **Figure 1.** Freeway junction A1/A4 in Gliwice (Poland).

*2.2. Source Materials*  When collecting source material for the research, two main criteria were taken into The development of road networks is one of the current symptoms of the interference in spatial structures of Gliwice. Two international corridors, the A1 and A4 highways, have become particularly

archives and libraries. Then, it was possible to collect maps of the selected area in the adopted scale. The period represented on the map constituted the second criteria adopted in the process of collecting materials. Particular states are separated by time intervals of 30–50 years. It occurred for a few collections that some specific sections, which were parts of the current city area, differed by several years, according to the dates on sheets. The maps from late '20s constituted the greatest problem, as not all sheets depicting the current area of Gliwice were prepared in accordance with land borders of that time. In such cases the Prussian topographic map of 1:25000, published at that

Furthermore, all the maps collected were included in the publishing series that covered the entire country (Figure 2). Having made the aforementioned assumption, one could select 6 map collections, starting from the second decade of 19th century, as the maps from that period constituted a significant step in making cartometric maps (based on constantly developed geodetic surveys and

The *Urmesstischblätter* map, prepared at the scale of 1:25 000, depicts the state of space since the 1820s. It was created as a result of the demand for a unified and accurate map of the entire Prussia after the decisions of the Congress of Vienna that introduced a new division of the European countries. To meet the demand, the network of triangulation stations was created. One could connect

consideration: the scale of the map and the period that the map presented. In terms of accuracy, the

relevant. The A4 has been visibly moved away from the center of Gliwice to the south, to the territory not so highly developed (including rural areas). The course of A1 is more complex, as the highway meanders between highly developed areas (Figure 1), which are connected with the specificity of the region, where, since the mid-19th century, the borders between neighboring towns and formerly independent settlements have been blurred as a result of the significant development of industry and settlements [12,13].

Apart from the city area, an extra buffer zone of 1 km exists, resulting from the location of the freeway junction near the border with Gierałtowice, meaning that certain freeway exits are situated fragmentarily out of the borders of Gliwice. Moreover, on the stretch of several kilometers, the A4 highway runs in the axis of the area border. The buffer adopted allowed one to present and analyze the studied phenomenon in a complex way.

#### *2.2. Source Materials*

When collecting source material for the research, two main criteria were taken into consideration: the scale of the map and the period that the map presented. In terms of accuracy, the scale of 1:25,000 was adopted, as it allowed one to distinguish between several categories of roads, at the same time presenting the state of space. In 19th century Gliwice was under Prussian partition, hence, it was necessary to run a search query of the cartographic resources from Polish and German archives and libraries. Then, it was possible to collect maps of the selected area in the adopted scale.

The period represented on the map constituted the second criteria adopted in the process of collecting materials. Particular states are separated by time intervals of 30–50 years. It occurred for a few collections that some specific sections, which were parts of the current city area, differed by several years, according to the dates on sheets. The maps from late '20s constituted the greatest problem, as not all sheets depicting the current area of Gliwice were prepared in accordance with land borders of that time. In such cases the Prussian topographic map of 1:25,000, published at that time, was used.

Furthermore, all the maps collected were included in the publishing series that covered the entire country (Figure 2). Having made the aforementioned assumption, one could select 6 map collections, starting from the second decade of 19th century, as the maps from that period constituted a significant step in making cartometric maps (based on constantly developed geodetic surveys and improved surveying instruments).

#### 2.2.1. Urmesstischblätter Map

The *Urmesstischblätter* map, prepared at the scale of 1:25,000, depicts the state of space since the 1820s. It was created as a result of the demand for a unified and accurate map of the entire Prussia after the decisions of the Congress of Vienna that introduced a new division of the European countries. To meet the demand, the network of triangulation stations was created. One could connect with the stations during field work by means of plane table surveying. Individual sheets were prepared according to instructions by units of soldiers and commanding officers, whose names were included at the bottom of each sheet [1]. In the 19th century, the area of Gliwice was under Prussian rule and such a situation continued till 1918. At that time, the Prussian topographic maps *Urmesstischblätter* and *Messtischblätter* were created. As Germany was united in the second half of the 19th century, the aforementioned maps presently constitute the heritage of the Federal Republic of Germany.

The map was prepared in Müffling polyhedric projection (*Preussische Polyederprojektion)*. For each sheet geographic coordinates in relation to Ferro Meridian were determined, adopting the sheet size of 100 (longitude) and 60 (latitude) [14,15]. The popular 19th century method of hachuring that allowed one to provide slope angles was applied to present the landform.

It is a manuscript map whose entire collection is a part of the Berlin State Library collection [16]. The sections for the area analyzed come from 1827 and 1828. The area in question consisted of 4 sheets with the following numbers: 3307, 3308, 3351 and 3352. There was no legend on the sheets, however, the legend was published separately in 1818 with detailed clarifications of multicolored symbols indicating a high informational potential of the map [17].

#### 2.2.2. Messtischblätter Map

published in several editions.

The 19th century progress in surveying methods and instruments was used by Prussian authorities in 1876 to prepare another topographic map. *Messtischblätter* maps, as a continuation of the previous map series, were also made at the scale of 1:25,000. A new series of surveys and adopted technical solutions allowed one to work out cartometric maps [10,18]. According to the name of the collection translated from German (messen—to measure/survey, Tisch—table), plane table surveying was still the basic method in use, however, more accurate instruments, such as a drawing board mounted on a tripod with a stadia, were also applied.

Maps were published by the method of printing (lithography), but in comparison with *Urmesstischblätter* the use of color was limited at that time. Monochromatic or dichromatic sheets were produced, with the blue color for marking surface water [19]. Maps were published until the '30 s and were updated a few times at that time. A reference to the Prime Meridian in Greenwich occurred in the description of coordinates on the sheet. *ISPRS Int. J. Geo-Inf.* **2020**, *9*, x FOR PEER REVIEW 5 of 14

**Figure 2.** Source materials for the six periods. **Figure 2.** Source materials for the six periods.

Two collections of the Prussian topographic maps used in the research share some common features, but also differ in some aspects. These are: the time of production, as *Urmesstischblätter* maps

however, inaccurate methods were still used on older maps. Thus, *Messtischblätter* maps showed higher measurement precision. The change in presenting the landform, apart from changes in the printing method and the use of colors, was also important. At the beginning, slopes of the land were presented through the method of hachuring, as contour lines were introduced in the second half of the 19th century. However, one should focus on the elements common to both types of maps, namely the same scale and invariable division into sheets. Maps were also characterized by a particular way of coding space used in Prussian topography. Separate legends for both series were produced and

2.2.3. Map of the Military Geographical Institute (Polish Wojskowy Instytut Geograficzny = WIG)

The late 1920s was another time period studied by the researchers. At that time, the Military Geographical Institute (English for: *Wojskowy Instytut Geograficzny = WIG*) was the institution responsible for the production and publication of maps. The institution was established after Poland had regained independence in 1918. Initially, its role was to collect maps produced at the time of

*Messtischblätter* maps retained the same sheet size and division into sections as the first series of maps from the early 19th century. Hence, the area of Gliwice occupied the same four sheets, however, the new numeration was introduced: 5677, 5678, 5777, 5778. The sheets presenting the state from the 1880s were mainly used in the research. What is more, the later *Messtischblätter* editions were used to complete lacking Polish maps from the late '20s. Some of the sheets have basic legends located beyond the frame and in some cases the sheets covering the area of Poland have descriptions in two languages (German and Polish).

Two collections of the Prussian topographic maps used in the research share some common features, but also differ in some aspects. These are: the time of production, as *Urmesstischblätter* maps present the state from the first half of the 19th century, whereas *Messtischblätter* maps emerged in 1876. Both map types were based on geodetic surveys (on previously measured triangulation networks), however, inaccurate methods were still used on older maps. Thus, *Messtischblätter* maps showed higher measurement precision. The change in presenting the landform, apart from changes in the printing method and the use of colors, was also important. At the beginning, slopes of the land were presented through the method of hachuring, as contour lines were introduced in the second half of the 19th century. However, one should focus on the elements common to both types of maps, namely the same scale and invariable division into sheets. Maps were also characterized by a particular way of coding space used in Prussian topography. Separate legends for both series were produced and published in several editions.

#### 2.2.3. Map of the Military Geographical Institute (Polish Wojskowy Instytut Geograficzny = WIG)

The late 1920s was another time period studied by the researchers. At that time, the Military Geographical Institute (English for: *Wojskowy Instytut Geograficzny* = *WIG*) was the institution responsible for the production and publication of maps. The institution was established after Poland had regained independence in 1918. Initially, its role was to collect maps produced at the time of partitions and copy them for the army. Then, the realization of triangulation work and the preparation of the new concept of topographic maps, used later as a basis for detailed maps made at the scale of 1:25,000, began. Maps were intended for the army, but they were also of great importance to economic and social needs [20].

The map was distinguished by the new system of topographic symbols (188 separations and 70 literal abbreviations). The map shared some common features with the Prussian *Messtischblätter* map in terms of the way of coding. The "Borowa Góra" coordinate system, along with a quasi-stereographic *WIG* projection, was adopted for the scale discussed. Those were monochromatic maps that sometimes used the brown color for the land form. The map produced by means of lithographic printing covered approximately 50% of the country's area [20].

According to indices, the modern area of Gliwice constitutes four sections of the map. Only two sheets covering the south-eastern and south-western part, made, respectively, in 1926 and 1933, were prepared. The remaining part of the area was a part of Germany at that time, therefore, two sheets presenting the state from 1928 and 1929 were completed with the *Messtischblätter* map. The WIG map had the basic legend with separations for roads, railroads and forests beyond the frame at the bottom of the sheet (Figure 3).

#### 2.2.4. The Topographic Map in the 1942 Coordinate System

After WW2, when Poland became a zone under the strong influence of the Soviet Union, cartography was divided into the civil and the military. Furthermore, maps were censored and access to them was strictly limited. In 1952, the Conference of Geodetic Service of USSR and People's Republic Countries was held. At the conference, the guidelines on map production, such as the 1942 coordinate system based on the Krasowski ellipsoid, and the division into sheets according to the International Map of the World 1:1,000,000, were adopted. States that participated in the conference, including Poland, were obliged to prepare a basic topographic map at the scale of 1:25,000 [21].

Maps used in the research, made according to the Gauss–Krüger coordinate system, come from the '60 s (EPSG 3330). They were published to meet economic needs and lacked coordinates. Their content, compared to military editions, was also limited and, in terms of topographic objects and land form, masked by some tricks (the information about confidentiality of the data was placed on the map's frame). Only after 1990 were confidentiality regulations lifted, and civil maps were finally published in this coordinate system [20].

The area researched encompasses two sheets (number 3 and 5) of the former Gliwice county. The maps lacked the information about the system of the symbols used (the legend), however, as the content was limited, and one could distinguish the quality and order of the material presented [22].

#### 2.2.5. The Topographic Map in the 1965 Coordinate System

The system adopted in 1968 was a result of the fact that the use of the 1942 system had been limited in civil cartography. Observing the global situation, i.e., the development of satellite programs, and running operations during the Cold War, the authorities in Moscow ordered the Eastern Bloc countries to prepare an independent system for producing economic maps. The above circumstances resulted in the preparation and publication of topographic maps at the scale of 1:25,000 and 1:50,000 since the late 1970 [21].

The Krasowski ellipsoid was the basic point of reference, but the system was not uniform for the entire country, as zones 1–4 were distinguished (prepared in the quasi-stereographic projection), and zone 5 was distinguished as well (made in the Gauss–Krüger coordinate system). The sheets had a different cut and division into sections in relation to the 1942 system. Moreover, maps in the 1965 system did not have the cartographic grid, so the topographic grid became the basis for the division into sections [20,21].

The current area of Gliwice is depicted on two map sheets in the 1965 system with 531.11 and 521.34 emblems in zone 5 (EPSG 2175), demonstrating the state of space from 1986. Furthermore, the limited content of the map was still censored and, in terms of graphic parameters, it was similar to military maps. An extended legend was placed on each sheet.

#### 2.2.6. OpenStreetMap (OSM)

Originally a project of the Internet community aimed at creating a free map of the entire globe since 2004, it was adopted as the updated version to be used [23] (EPSG 3857). The OSM base map, as a topographic map, was used to create this article because it was up-to-date and compatible with the web technology used in the web programming stage. The service provides many opportunities, such as visible legend (the "i" button on the website). Downloadable OSM data are saved in a PostgreSQL relational database, without spatial extensions, in the WGS84 coordinate system. Roads constitute 28% of the data [24]. It is possible to display data in the 21-level zoom. The largest available scale: 1:500 (zoom 20), the smallest: 1:500,000,000 (zoom 0).

#### *2.3. Selection of Objects from the Legend*

Given that map series have several editions with symbol explanations, the query of maps and their legends in terms of the aspects studied (major roads) made the authors adopt basic legend editions (Figure 3). The authors adopted the explanations of symbols from map marginalia (if they occurred, e.g., WIG maps, the 1965 system) or, for *Urmesstischblätter* maps, the first edition of the legend, as it was adequate for the content of the sheets collected. Furthermore, the later, usually more comprehensive editions of legends did not demonstrate any changes in the way major road categories were distinguished. *Messtischblätter* maps' legends included highways in later editions.

In the research, legends constituted a starting point and a point of reference that allowed the authors to identify given road categories on maps. However, it was the analysis of every single sheet that provided the information about the category of roads that occurred there. On the maps analyzed, the category of the same road stretches would frequently change, depending on the time period the

*2.3. Selection of Objects from the Legend*

2.2.6. OpenStreetMap (OSM)

map came from. It could result from different rules of categorization or technical guidelines used for making maps, as well as from road rebuilding/expansion. Using legends, the authors followed the changes in the evolution of major road corridors in Gliwice. the category of the same road stretches would frequently change, depending on the time period the map came from. It could result from different rules of categorization or technical guidelines used for making maps, as well as from road rebuilding/expansion. Using legends, the authors followed the

that provided the information about the category of roads that occurred there. On the maps analyzed,

were distinguished. *Messtischblätter* maps' legends included highways in later editions.

*ISPRS Int. J. Geo-Inf.* **2020**, *9*, x FOR PEER REVIEW 7 of 14

limited content of the map was still censored and, in terms of graphic parameters, it was similar to

Originally a project of the Internet community aimed at creating a free map of the entire globe since 2004, it was adopted as the updated version to be used [23] (EPSG 3857). The OSM base map, as a topographic map, was used to create this article because it was up-to-date and compatible with the web technology used in the web programming stage. The service provides many opportunities, such as visible legend (the "i" button on the website). Downloadable OSM data are saved in a PostgreSQL relational database, without spatial extensions, in the WGS84 coordinate system. Roads constitute 28% of the data [24]. It is possible to display data in the 21-level zoom. The largest available

Given that map series have several editions with symbol explanations, the query of maps and their legends in terms of the aspects studied (major roads) made the authors adopt basic legend editions (Figure 3). The authors adopted the explanations of symbols from map marginalia (if they occurred, e.g., WIG maps, the 1965 system) or, for *Urmesstischblätter* maps, the first edition of the legend, as it was adequate for the content of the sheets collected. Furthermore, the later, usually more comprehensive editions of legends did not demonstrate any changes in the way major road categories

military maps. An extended legend was placed on each sheet.

scale: 1:500 (zoom 20), the smallest: 1:500000000 (zoom 0).

Figure 3 presents some fragments of legends that show the ways of presenting roads of the highest category for the six maps analyzed. All of the legends depict roads, starting from the highest category. One can observe a similar way of coding these objects in the editions researched, namely the use of bold black contour on both sides of the line. Additionally, in full-color editions, the extra filling color was added (e.g., *Urmesstischblätter*, *OSM*). Explanations of symbols for the topographic map in the 1942 coordinate system constituted an exception among the materials collected. They came from the edition that was published many years after the map, as earlier legends, reserved for military map publications, had not been available. changes in the evolution of major road corridors in Gliwice. Figure 3 presents some fragments of legends that show the ways of presenting roads of the highest category for the six maps analyzed. All of the legends depict roads, starting from the highest category. One can observe a similar way of coding these objects in the editions researched, namely the use of bold black contour on both sides of the line. Additionally, in full-color editions, the extra filling color was added (e.g., *Urmesstischblätter*, *OSM*). Explanations of symbols for the topographic map in the 1942 coordinate system constituted an exception among the materials collected. They came from the edition that was published many years after the map, as earlier legends, reserved for military map publications, had not been available.

**Figure 3.** Ways of presenting the highest category of roads in the legends of the six maps. **Figure 3.** Ways of presenting the highest category of roads in the legends of the six maps.

#### *2.4. Gathering and Processing Data*

After six time periods demonstrating spatial structures with the road network of the city had been selected for further processing, the authors applied the scheme they had developed in previous studies [10]. In this stage of research, the Qgis 3.12 program was used. Maps were registered to the current WGS-84 coordinate system (EPSG 4326). The registration was made by means of affine transformation based on 4 ground control points [3]. Due to incomplete cartometricity, the georeferencing of the oldest map required particular care. In order to achieve optimum results (low RMSE with the appropriate proportion of a sheet), the larger number of ground control points was adopted. Selected fragments of *Urmesstischblätter* sheets that were located in Gliwice or nearby were also registered. The registration of *Urmesstischblätter* maps was the subject of the previous research [17,25].

Then, according to the scheme adopted, stretches of roads of the highest category, occurring on given sheets, were vectorized. On the OSM map, suitable fragments of linear objects were selected. Vectorization of roads according to the trace was carried out, starting from more recent to older maps and highlighting objects previously selected on the basis of legends. Roads from each period were saved in separate layers in the .shp format and finally six levels presenting the system of major roads in given time periods were gained [10].

#### *2.5. Web Map Programming*

Roads representing the evolution of European road corridors (the A1 and A4 highways) for the six maps analyzed, prepared in the process of vectorization, constituted a basis for the web map. In the process of programming the interactive web map, the Leaflet library was used. As a JavaScript library, Leaflet is efficient, useful and open-source [9,10,26]. It was also widely used in similar map versions. Leaflet supports the following extensions: GeoJSON, TopoJSON and KML, therefore, vectors of roads saved as .shp extension files (and other, i.e., .dbf, .shx, .cpg, .prj) had to be converted to GeoJSON files [27]. According to Horbi´nski and Lorek [10], it is recommended to use this extension for files of small size and representing a relatively small area. Furthermore, GeoJSON is a frequently adopted standard in preparing layers of interactive web maps [28]. Necessary conversion, like in previous stages, was done in QGIS 3.12.

Having converted layers, researchers had to create the interactive web map on the basis of the previous assumptions [29]. According to the assumptions, the interactive web map presenting the evolution of major roads in Gliwice and construction of the junction of the European corridors (the A1 and A4 highways) is going to consist of the main map, overview map, layers and scale (Figure 4). In addition to the layers presenting the state of the highest class roads, the authors added layers of the boundaries of the Gliwice poviat and layers with the most important road intersections in a specific period of time.

To design the interactive web map (main map), the basic functions of Leaflet and two plugins were used (Leaflet Panel Layers [30] and leaflet-graphicscale [31]). The first one allowed the user to switch road layers on and off in the six stages analyzed. The other one represented map scales graphically. The overview map is an additional element. The overview map is an interactive map presenting the entire course of the roads. Created as a separate object, it does not have any links with the main map. The interactive web map (Figure 4) is available here: http://kartografia.amu.edu.pl/Motorway/index. html, on the server of Adam Mickiewicz University in Pozna ´n. *ISPRS Int. J. Geo-Inf.* **2020**, *9*, x FOR PEER REVIEW 9 of 14

**Figure 4.** Interactive web map of the development of the European freeway junction A1/A4. **Figure 4.** Interactive web map of the development of the European freeway junction A1/A4.

#### **3. Results and Discussion 3. Results and Discussion**

Union) [36].

Over a span of nearly two centuries, one can observe changes in the way major roads run. On the interactive web map, the occurrence of the junctions of these roads in given time periods were marked with the sight, starting with the situation when they connected in the center of Gliwice (the state from 1828). According to Antrop [32], it was one of the models typical of the settlement network of that time. The situation changed on the maps published nearly 100 years later. A crossing of major roads occurred in areas moved northwards from the city center. The change was linked with the urbanization and suburbanization process and intensified traffic that forced the construction of bypasses and moving the traffic away from the center [12]. A construction of the freeway on the outskirts of the city and moving the main road junction to the south-east was the next stage. Over a span of nearly two centuries, one can observe changes in the way major roads run. On the interactive web map, the occurrence of the junctions of these roads in given time periods were marked with the sight, starting with the situation when they connected in the center of Gliwice (the state from 1828). According to Antrop [32], it was one of the models typical of the settlement network of that time. The situation changed on the maps published nearly 100 years later. A crossing of major roads occurred in areas moved northwards from the city center. The change was linked with the urbanization and suburbanization process and intensified traffic that forced the construction of bypasses and moving the traffic away from the center [12]. A construction of the freeway on the outskirts of the city and moving the main road junction to the south-east was the next stage.

The A4 freeway is an extension of the east–west freeway running through Germany (*Autobahn 4)*. Such roads were built there in the 1930s as a reply to a visible increase in the number of cars [37]. A short stretch of freeway was also constructed in Poland at that time. The A1 freeway ("Amber Highway") was constructed in the 1960s as a part of the project whose aim was to connect the north with the south of Europe. The name and course of the freeway refer to the historical Amber Road connecting the Baltic with the Mediterranean Sea. On the border with the Czech Republic, it connects

The "Gliwice Sośnica" junction was constructed between 2008 and 2010 and connected three thoroughfares on three separate levels. The A4 highway runs the lowest, the route 44 runs above and the A4 highway is the highest. Building was planned in a place where the Route 44 and the A4 highway crossed in 2005. Building one of the largest European freeway junctions in Gliwice ended in late 2009 and since September 2011 all sidings have functioned [39]. In the junction, two trans-

with the D1 freeway (*dálnice D1*), dating back to the 1930s [38].

development [34]. Considering this part of Europe, it becomes clearly visible that the city of Gliwice is located on the crossing of two corridors that were developing gradually in European states to finally make a network of the most significant roads on the continent [35]. Currently, both freeways are a part of the Trans-European Transport Network that makes a coherent transport network on the entire continent. The idea of the network emerged and was adopted in the 1990s as a result of international conferences (decisions of the European Parliament and the Council of the European

The level of industrialization and urbanization of the city and its neighborhood in given time periods determined the expansion of road network, including thoroughfares of the highest category [33]. The current state of the road network shows that mechanisms pushing the development were

The level of industrialization and urbanization of the city and its neighborhood in given time periods determined the expansion of road network, including thoroughfares of the highest category [33]. The current state of the road network shows that mechanisms pushing the development were not only of the bottom-up character and were not resulting only from local needs in terms of economic development [34]. Considering this part of Europe, it becomes clearly visible that the city of Gliwice is located on the crossing of two corridors that were developing gradually in European states to finally make a network of the most significant roads on the continent [35]. Currently, both freeways are a part of the Trans-European Transport Network that makes a coherent transport network on the entire continent. The idea of the network emerged and was adopted in the 1990s as a result of international conferences (decisions of the European Parliament and the Council of the European Union) [36].

The A4 freeway is an extension of the east–west freeway running through Germany (*Autobahn 4*). Such roads were built there in the 1930s as a reply to a visible increase in the number of cars [37]. A short stretch of freeway was also constructed in Poland at that time. The A1 freeway ("Amber Highway") was constructed in the 1960s as a part of the project whose aim was to connect the north with the south of Europe. The name and course of the freeway refer to the historical Amber Road connecting the Baltic with the Mediterranean Sea. On the border with the Czech Republic, it connects with the D1 freeway (*dálnice D1*), dating back to the 1930s [38].

The "Gliwice So´snica" junction was constructed between 2008 and 2010 and connected three thoroughfares on three separate levels. The A4 highway runs the lowest, the route 44 runs above and the A4 highway is the highest. Building was planned in a place where the Route 44 and the A4 highway crossed in 2005. Building one of the largest European freeway junctions in Gliwice ended in late 2009 and since September 2011 all sidings have functioned [39]. In the junction, two trans-European transport corridors were linked. The A4 constitutes a part of the international E40 road, leading from Ostend to Kiev, crossing the Polish–German border in J˛edrzychowice and the Polish–Ukrainian border in Korczowa in eastern Poland. The M10 freeway leading to Lviv is supposed to be a continuation of the corridor on the Ukrainian side. The A1 highway constitutes a part of the international E75 north–south road. This corridor connects Norway with Greece and in Poland, when finished, is going to run from Gda ´nsk through Katowice to the border with the Czech Republic [40].

Table 1 contains information about the changes in the total length of major roads. The results come from calculating vector data presented on the maps from particular time periods, considering also the margin of 1 km around Gliwice. When comparing the data from the table, one needs to note that they result from the way particular stretches of roads were classified on different maps, which means that some of the roads may have been classified differently on particular maps, their rebuilding or modernization being actually not considered. Nevertheless, the above data juxtaposed with the interactive map present the gist of the changes occurring.

Among the six time periods researched, the greatest increase in major roads took place in the 19th century, reflecting changes occurring as a result of the industrial revolution (and also other factors). Out of all the periods studied, this was the longest one (1828–1880). At that time, the number of major roads increased more than twice. The processes initiated then continued for the following decades, as demonstrated on the map from 1928. A significant change on the Messtischblätter map was that the roads connecting north-west and south-west with south-east became officially recognized as major roads.

Another major change occurred between 1928 and 1960. The maps depicted the state before and after WW2. Generally, the road network became more dense and the increase in length of major roads (by approximately 40%) was connected with rebuilding the area after the war. Moreover, some projects are already being carried out to create new connections, such as constructing a bypass in the northern part to avoid passing through the city center.

The slightest changes are demonstrated on the map from 1960 and 1986. That was the shortest period of all. The web map shows that there was a balance between the number of roads that were constructed and those that disappeared. When calculating the total length of major roads for both map series, it is necessary to consider the issue of censorship and changes in the way particular road stretches were classified.

A highly significant change, the greatest one since the transformation of the 19th century, took place between the last two periods. The construction of freeways significantly impacted the road landscape of the region. New long corridors that run through Gliwice, connecting west with east and north with south, are the most relevant roads. Furthermore, they are completed by the northern bypass constructed in the previous decades, along with several road stretches marked as major, connected with freeways by means of junctions.

**Table 1.** The length of the highest category of road networks.

In order to collect data on changes occurring in space, historical maps are used and compiled with modern cartographic sources by means of new technologies [41–43]. Reconstructing the structure of historical road networks and the interactive way of presenting data help the authors develop the methodologies for retrospective studies [44–46]. The use of maps from many periods makes it possible to follow the course of changes taking place [47] in terms of the road network and the location of main junctions.

The approach adopted in the research develops previous studies by adding the new way of data presentation. Thematic visualizations in the form of web maps increase the availability of the information about the historical states of spaces. The web map may constitute a research material concerning historical cartography for all intranet users, including non-experts. Users receive the finished product from properly selected (in terms of scale and time periods), obtained and processed archival cartographic sources.

Each stage, from selecting the archival map to converting its content to the interactive form, is connected with work on the methodology of using historical maps in the research [17,48,49] or proper registration [25,50,51]. The access to historical maps (particularly to the editions from the early 19th century) is frequently difficult and the opportunity to publish archival materials is often limited. It is also important to collect data on the circumstances in which individual archival maps were published. Adopting a uniform scale for all the cartographic materials obtained allows one to compare data with similar accuracy, considering the specificity of each series (e.g., the subjectivity of topographers making maps in the first half of the 19th century [1] or censorship of the maps from the second half of the 20th century) [20].

According to the cartography cube theory [52], adding interactivity to spatial data obtained from the scans of archival maps boosts the potential of these maps for retrospective studies (obtaining new knowledge about the structure of the environment in the past). Such actions raise the status of paper maps in the cube from the level of data presentation to the level of research.

#### **4. Conclusions**

In comparison with other sources of information about space, such as descriptions, photos, drafts or projects, the map demonstrates a given area in a comprehensive way, along with its elements (objects), spatial layout, and the relations between them. Individual states of the evolving space were presented on the topographic maps used. The juxtaposition between selected objects in the form of vector layers of major roads from the periods following shows that the current area of Gliwice is characterized by the transformation of postindustrial areas' infrastructure and the change in space use in terms of the construction of thoroughfares and international freeway junctions [25,32,53].

Summing up the way of demonstrating changes that occurred in space in the example of the European freeway A1/A4 junction, the authors corroborate the hypothesis from this article's introduction. The interactive map that was designed proves that there are opportunities to reconstruct historical states of space, by means of modern multimedia technologies and cartographic sources. Creating interactive thematic web maps that show transformations of selected objects or spatial relations is possible thanks to the use of the approach presented. In addition, there is a wide range of opportunities provided by multimedia cartography in terms of presenting the data collected. The road layers placed on the interactive web map can be extended by adding other available multimedia, such as photos or even videos [25]. According to the authors, the use of the interactive web map is the most comprehensive method of all technologies used by modern cartography. Thanks to the interactive web map, the user has all the data collected in one place. Transparency in the reception of information is also guaranteed, as layers can be turned on and off. The interactive web map presented by the authors creates no information overflow effect [54,55], which may result in its better use [23]. Spatial data collected from different cartographic publications (from the first half of the 19th century till the present) consider the most significant aspects of changes in the road network of the area analyzed in a detailed and user-friendly way.

The authors considered the use of other archival cartographic publications that could make the research more accurate (by providing shorter time periods and more detailed maps), however, the adopted scale of 1:25,000 and time spans of 30–50 years, resulting from the previously executed query, provided the optimal juxtaposition of sources that allowed the authors to present spatial changes in the network of the most significant roads.

Moreover, the authors note the opportunity to increase the interactivity of the map created by introducing additional information with a mouse over or a click on the layers. In their interactive web map, as an example of placing information in a pop-up, they included information about the source map for each of the road layers. Opportunities to add new functions, linked with the development of the library that was used in creating the interactive web map or resulting from technological advancement (i.e., improvement of the software, updated search engines, implementation of the new W3C standards) were also considered [10]. Choosing layers independently and comparing them with one another would also be an interesting option. The layer selection order sets the display order, which allows the user to compare older layers to more recent layers and vice versa.

The above method of data presentation optimizes the possibility of interpreting selected thematic issues, among others in the field of transformation of thoroughfares. The aspect of evolution of the main thoroughfares and the interactive way of presenting it may be used for researching other significant places in Europe, e.g., to compare the level of land transformation resulting from the expansion of freeway junctions.

**Author Contributions:** Conceptualization, D.L.; Data curation, T.H.; Formal analysis, T.H.; Methodology, D.L.; Project administration, D.L.; Resources, D.L.; Software, T.H.; Visualization, T.H.; Writing—original draft, D.L. and T.H.; Writing—review & editing, D.L. and T.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This paper is the result of research on visualization methods carried out within statutory research in the Department of Cartography and Geomatics, Faculty of Geographical and Geological Sciences, Adam Mickiewicz University in Pozna ´n, in Poland.

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

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**5.1. Intracellular enzymes activity in type 1 diabetes**

A number of natural antioxidants are present in the body to scavenge oxygen free radicals and prevent oxidative damage to biological membranes. Antioxidant defense mechanisms involve both non-enzymatic and enzymatic strategies. One group of these antioxidants is in‐ tracellular enzymes such as manganese superoxide dismutase, catalase, glutathione peroxi‐ dase, and glutathione-S-transferases. These enzymes represent a protective mechanism against the damage caused by the oxidative stress and most of these enzymes are polymor‐ phic (Fang et al., 2002; Mates et al., 1999).

et al., 1995). The low glutathione peroxidase activity could be directly explained by either low glutathione content or enzyme inactivation under sever oxidative stress (Faure et al., 1995). However, some authors found no differences between glutathione peroxidase activity of type 1 diabetic patients and control subjects (Jain et al., 1994; Murakami et al., 1993;

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Catalase, located in peroxisomes, decomposes hydrogen peroxide to water and oxygen (Win‐ terbourn & Metodiewa, 1994). In addition, glutathione peroxidase in the mitochondria and the lysosomes also catalyses the conversion of hydrogen peroxide to water and oxygen (Yung et al., 2006). A significant increase in the catalase activity in lymphocytes was found in 40 chil‐ dren with type 1 diabetes during all phases (at the beginning of diabetes, in remission period and in the later chronic course) compared with the control group. The highest catalase activi‐ ty occurs in the early course of disease followed by a linear decrease and the lowest activity in chronic course (Zivić, 2008). Conversely Dave and colleagues (2007) reported significant de‐ creased glutathione peroxidase, catalase and glutathione, and significant increase in thiobar‐ bituric acid reactive substances concentration in type 1 diabetic patients with and without

In addition to enzymatic antioxidants, the major natural antioxidants, most of which are de‐ rived from dietary sources are vitamin A, vitamin C or ascorbic acid, vitamin E and carote‐ noids. Water-soluble vitamin C and fat-soluble vitamin E together make up the antioxidant system for mammalian cells (Engler et al., 2003). Vitamins A, C, and E are obtained from the diet and function to directly detoxify free radicals. Vitamin C forms the first line of defense against plasma lipid peroxidation is considered the most important antioxidant in plasma (Frei et al., 1990). Vitamin C under certain conditions may foster toxicity by generating prooxidants, and is also engaged in the recycling processes which involved the generation of reduced forms of the vitamins. In the processes of regeneration, α-tocopherol is reconstitut‐ ed when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is

Vitamin E involves all tocopherol and tocotrienol derivatives that comprise the major lipo‐ philic exogenous antioxidant in tissues (Di Mambro et al., 2003). Vitamin E, a component of the total peroxyl radical-trapping antioxidant system reacts directly with superoxide and peroxyl radicals, and singlet oxygen and in so doing protects membranes from lipid peroxi‐ dation (Weber & Bendich, 1997). In a study by Gupta et al. 2011 that evaluated the oxidative stress in 20 type 1 diabetic children, reduced glutathione and vitamin E levels were de‐ creased and malondialdehyde levels were elevated compared with controls. After supple‐ mentation with vitamin E (600 mg/daily for three months) there was a significant decrease in malondialdehyde levels and significant increase in glutathione and vitamin E. The find‐ ings indicate that vitamin E ameliorates oxidative stress in type 1 diabetes mellitus patients and improves antioxidant defense system. In a latter study high-dose vitamin E supplemen‐ tation (1200 mg/day) reduces markers of oxidative stress and improves antioxidant defense

nephropathy compared with normal healthy individuals (Dave et al., 2007).

**5.2. Non-enzymatic antioxidant levels in type 1 diabetes**

formed, is recycled by glutathione (Weber, 1997).

Majchrzak et al., 2001).

Superoxide dismutase is considered a primary enzyme since it is involved in the direct elim‐ ination of reactive oxygen synthase (Halliwell, 1994). Isoforms of superoxide dismutase are Cu/Zn-superoxide dismutase which is found in both the cytoplasm and the nucleus, and Mn-superoxide dismutase that is present in the mitochondria. The latter can be released into extracellular space (Reiter et al., 2000). Cu/Zn-superoxide dismutase over-expression inhib‐ its oxidized low density lipoprotein which is can elevate deoxyribonucleic acid binding ac‐ tivity of activator protein-1 and NF-κB (Yung et al., 2006). Superoxide dismutase catalyzes the conversion of superoxide anion radicals produced in the body to hydrogen peroxide. This decreases the possibility of superoxide anion interacting with nitric oxide to form reac‐ tive peroxynitrite (Reiter et al., 2000). Low Cu/Zn-superoxide dismutase is a potential early marker of susceptibility to diabetic vascular disease. Suys et al. (2007) found that erythrocyte superoxide dismutase activity and Cu/Zn-superoxide dismutase were higher in type 1 dia‐ betic subjects and was positively associated with flow-mediated dilatation. Based on these findings the authors suggest that higher circulating Cu/Zn-superoxide dismutase could pro‐ tect type 1 diabetic children and adolescents against endothelial dysfunction (Suys et al., 2007). Furthermore, Reznick and colleagues analyzed both serum and salivary superoxide dismutase activity in 20 patients with type 1 diabetes mellitus. A significant association was found between the level of glycemic control as indicated by the glycated hemoglobin values and an increase in both salivary and serum superoxide dismutase activity (Reznick et al., 2006). On the contrary, in a study which assessed correlations between increase of oxidative stress and the development of microalbuminuria in 87 type 1 diabetic patients (44 with nor‐ mal urinary protein excretion, and 43 with microalbuminuria), there was a decreased in ac‐ tivity of superoxide dismutase. This was associated with an increased microalbuminuria in type 1 diabetic patients (Artenie et al., 2005).

Selenium-dependent glutathione peroxidase works in conjunction with superoxide dismu‐ tase in protecting cell proteins and membranes against oxidative damage. In the literature, glutathione peroxidase response to diabetes has been conflicting. Diabetics have been re‐ ported to be associated with increased glutathione peroxidase activity in 90 pregnant wom‐ en with type 1 diabetes mellitus (Djordjevic et al., 2004) and in young diabetic patients (Ndahimana et al., 1996). On the other hand, decreased glutathione peroxidase activity was reported in the early stages of type 1 diabetes in children and adolescents (Dominguez et al., 1998) or unchanged in type 1 diabetic patients with early retina degenerative lesions (Faure et al., 1995). The low glutathione peroxidase activity could be directly explained by either low glutathione content or enzyme inactivation under sever oxidative stress (Faure et al., 1995). However, some authors found no differences between glutathione peroxidase activity of type 1 diabetic patients and control subjects (Jain et al., 1994; Murakami et al., 1993; Majchrzak et al., 2001).

Catalase, located in peroxisomes, decomposes hydrogen peroxide to water and oxygen (Win‐ terbourn & Metodiewa, 1994). In addition, glutathione peroxidase in the mitochondria and the lysosomes also catalyses the conversion of hydrogen peroxide to water and oxygen (Yung et al., 2006). A significant increase in the catalase activity in lymphocytes was found in 40 chil‐ dren with type 1 diabetes during all phases (at the beginning of diabetes, in remission period and in the later chronic course) compared with the control group. The highest catalase activi‐ ty occurs in the early course of disease followed by a linear decrease and the lowest activity in chronic course (Zivić, 2008). Conversely Dave and colleagues (2007) reported significant de‐ creased glutathione peroxidase, catalase and glutathione, and significant increase in thiobar‐ bituric acid reactive substances concentration in type 1 diabetic patients with and without nephropathy compared with normal healthy individuals (Dave et al., 2007).

### **5.2. Non-enzymatic antioxidant levels in type 1 diabetes**

**5. Enzymatics and non-enzymatic antioxidants**

A number of natural antioxidants are present in the body to scavenge oxygen free radicals and prevent oxidative damage to biological membranes. Antioxidant defense mechanisms involve both non-enzymatic and enzymatic strategies. One group of these antioxidants is in‐ tracellular enzymes such as manganese superoxide dismutase, catalase, glutathione peroxi‐ dase, and glutathione-S-transferases. These enzymes represent a protective mechanism against the damage caused by the oxidative stress and most of these enzymes are polymor‐

Superoxide dismutase is considered a primary enzyme since it is involved in the direct elim‐ ination of reactive oxygen synthase (Halliwell, 1994). Isoforms of superoxide dismutase are Cu/Zn-superoxide dismutase which is found in both the cytoplasm and the nucleus, and Mn-superoxide dismutase that is present in the mitochondria. The latter can be released into extracellular space (Reiter et al., 2000). Cu/Zn-superoxide dismutase over-expression inhib‐ its oxidized low density lipoprotein which is can elevate deoxyribonucleic acid binding ac‐ tivity of activator protein-1 and NF-κB (Yung et al., 2006). Superoxide dismutase catalyzes the conversion of superoxide anion radicals produced in the body to hydrogen peroxide. This decreases the possibility of superoxide anion interacting with nitric oxide to form reac‐ tive peroxynitrite (Reiter et al., 2000). Low Cu/Zn-superoxide dismutase is a potential early marker of susceptibility to diabetic vascular disease. Suys et al. (2007) found that erythrocyte superoxide dismutase activity and Cu/Zn-superoxide dismutase were higher in type 1 dia‐ betic subjects and was positively associated with flow-mediated dilatation. Based on these findings the authors suggest that higher circulating Cu/Zn-superoxide dismutase could pro‐ tect type 1 diabetic children and adolescents against endothelial dysfunction (Suys et al., 2007). Furthermore, Reznick and colleagues analyzed both serum and salivary superoxide dismutase activity in 20 patients with type 1 diabetes mellitus. A significant association was found between the level of glycemic control as indicated by the glycated hemoglobin values and an increase in both salivary and serum superoxide dismutase activity (Reznick et al., 2006). On the contrary, in a study which assessed correlations between increase of oxidative stress and the development of microalbuminuria in 87 type 1 diabetic patients (44 with nor‐ mal urinary protein excretion, and 43 with microalbuminuria), there was a decreased in ac‐ tivity of superoxide dismutase. This was associated with an increased microalbuminuria in

Selenium-dependent glutathione peroxidase works in conjunction with superoxide dismu‐ tase in protecting cell proteins and membranes against oxidative damage. In the literature, glutathione peroxidase response to diabetes has been conflicting. Diabetics have been re‐ ported to be associated with increased glutathione peroxidase activity in 90 pregnant wom‐ en with type 1 diabetes mellitus (Djordjevic et al., 2004) and in young diabetic patients (Ndahimana et al., 1996). On the other hand, decreased glutathione peroxidase activity was reported in the early stages of type 1 diabetes in children and adolescents (Dominguez et al., 1998) or unchanged in type 1 diabetic patients with early retina degenerative lesions (Faure

**5.1. Intracellular enzymes activity in type 1 diabetes**

phic (Fang et al., 2002; Mates et al., 1999).

230 Type 1 Diabetes

type 1 diabetic patients (Artenie et al., 2005).

In addition to enzymatic antioxidants, the major natural antioxidants, most of which are de‐ rived from dietary sources are vitamin A, vitamin C or ascorbic acid, vitamin E and carote‐ noids. Water-soluble vitamin C and fat-soluble vitamin E together make up the antioxidant system for mammalian cells (Engler et al., 2003). Vitamins A, C, and E are obtained from the diet and function to directly detoxify free radicals. Vitamin C forms the first line of defense against plasma lipid peroxidation is considered the most important antioxidant in plasma (Frei et al., 1990). Vitamin C under certain conditions may foster toxicity by generating prooxidants, and is also engaged in the recycling processes which involved the generation of reduced forms of the vitamins. In the processes of regeneration, α-tocopherol is reconstitut‐ ed when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is formed, is recycled by glutathione (Weber, 1997).

Vitamin E involves all tocopherol and tocotrienol derivatives that comprise the major lipo‐ philic exogenous antioxidant in tissues (Di Mambro et al., 2003). Vitamin E, a component of the total peroxyl radical-trapping antioxidant system reacts directly with superoxide and peroxyl radicals, and singlet oxygen and in so doing protects membranes from lipid peroxi‐ dation (Weber & Bendich, 1997). In a study by Gupta et al. 2011 that evaluated the oxidative stress in 20 type 1 diabetic children, reduced glutathione and vitamin E levels were de‐ creased and malondialdehyde levels were elevated compared with controls. After supple‐ mentation with vitamin E (600 mg/daily for three months) there was a significant decrease in malondialdehyde levels and significant increase in glutathione and vitamin E. The find‐ ings indicate that vitamin E ameliorates oxidative stress in type 1 diabetes mellitus patients and improves antioxidant defense system. In a latter study high-dose vitamin E supplemen‐ tation (1200 mg/day) reduces markers of oxidative stress and improves antioxidant defense in young patients with type 1 diabetes mellitus. However vitamin E supplementation did not decreased albumin excretion rate in these patients (Giannini et al., 2007).

tion. At low physiological oxygen pressures, it exhibits effective radical-trapping antioxidant behaviour (Frei, 1994). Coenzyme Q10 has been found to have a very important role in mitochondrial bioenergetics. It is an electron carrier-proton translocator in the respi‐ ratory chain and potent antioxidant which works by directly scavenging radicals or indirect‐ ly by regenerating vitamin E. In a study by Menke and colleagues (2008), plasma concentrations of coenzyme Q10 in 39 children with type 1 diabetes mellitus were higher than in healthy children. The findings suggest that elevated plasma concentration and the intracellular redox capacity of coenzyme Q10 in diabetic children may contribute to the body's self-protection during a state of enhanced oxidative stress (Menke et al., 2008). In an‐ other study, Salardi and colleagues (2004) determine whether serum hydroperoxides as oxi‐ dative markers and vitamin E and coenzyme Q10 as indexes of antioxidant capacity could be related to metabolic control in 75 unselected children, adolescents, and young adults with type 1 diabetes. Vitamin E and coenzyme Q10 were not significantly different from agematched control subjects. However, there were significant positive correlations between coenzyme Q10 and glycated hemoglobin, and vitamin E and glycated hemoglobin. It was al‐ so observed that diabetic patients with poor metabolic control and complications had elevat‐

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Small molecules that have antioxidant capacity such as glutathione and uric acid are synthe‐ sized or produced within the body (Engler et al., 2003). A study by Maxwell et al. (1997) found significantly reduced total serum antioxidant status in 28 patients with type 1 diabe‐ tes mellitus as attributed by lower uric acid and vitamin C levels. Furthermore, multiple re‐ gression analysis showed that uric acid, vitamin E and vitamin C were the main

Oxidative stress and its contribution to low-density lipoprotein oxidation have been impli‐ cated in the pathogenesis of vascular diabetic complications. Diabetes produces disturban‐ ces of lipid profiles, especially an increased susceptibility to lipid peroxidation, which is responsible for increased incidence of atherosclerosis, a major complication of diabetes mel‐ litus (Siu & To, 2002). Polyunsaturated fatty acids with multiple bonds and lipoproteins in the plasma membrane are very susceptible to attack by reactive oxygen species (Esterbauer & Schaur, 1991). The hydroxyl radicals extract a hydrogen atom from one of the carbon atoms in the polyunsaturated fatty acid and lipoproteins, initiating a free radical chain reac‐ tion which leads to lipid peroxidation. This characterized by membrane protein damage through subsequent free radical attacks (Halliwell, 1995). Lipid peroxidation can produce advanced products of oxidation, such as aldehydes, alkanes and isoprostanes (Moore & Roberts, 1998). Elevation of lipid peroxidation negatively affects membrane function causing reduced membrane fluidity and changing the activity of membrane bound enzymes and re‐

ed vitamin E levels and coenzyme Q10 levels (Salardi et al., 2004).

contributors to serum total antioxidant activity.

ceptors (Acworth et al., 1997).

**6. Markers of oxidative stress in type 1 diabetes**

**6.1. Biomarkers of lipid peroxidation in type 1 diabetes**

α-Tocopherol is very effective in lipid peroxidation inhibition and is the primary *in vivo* chain-breaking, lipid-soluble antioxidant in human serum. A reduction in serum α-toco‐ pherol could be attributed to its consumption while scavenging free radicals in lipoproteins or biomembranes (Frei, 1994). In the Pittsburgh Epidemiology of Diabetes Complications Study cohort, a 10-year prospective study of childhood-onset type 1 diabetes, α-tocopherol or γ-tocopherol did not showed protection against incident coronary artery disease overall. However, high α-tocopherol levels among patients with renal disease and in those using vi‐ tamin supplements were associated with lower coronary artery disease risk in type 1 diabe‐ tes (Costacou et al., 2006). All the antioxidants work in a synergistic manner with each other and against different types of free radicals. This is shown in the way in which vitamin E suppresses the propagation of lipid peroxidation, and vitamin C working with vitamin E in‐ hibits hydroperoxide formation (Laight et al., 2000).

Glutathione functions as a direct free-radical scavenger, and as a co-substrate for glutathione peroxidase activity (Meister & Anderson, 1983). Glutathione, a tri-peptide present in milli‐ molar concentrations is the most prevalent low-molecular weight peptide antioxidant in cells. Reduced glutathione normally plays the role of a direct intracellular free-radical scav‐ enger through interaction with free radicals and is the substrate of many xenobiotic elimina‐ tion reactions (Gregus et al., 1996). It is also involved in other cellular functions such as the elimination of hydrogen peroxide, detoxification processes such as protection of the sulf‐ hydryl group of cysteine in proteins, and regeneration of oxidized vitamin E (Lu, 1999). In 30 children with type 1 diabetes at onset, there was a significant reduction in all glutathione forms (total, reduced, oxidized, and protein-bound glutathione). This indicates that there is glutathione depletion upon early onset of type 1 diabetes mellitus (Pastore et al., 2012). In another study, Likidlilid et al. (2007) compared the glutathione level, and glutathione perox‐ idase activity in 20 type 1 diabetic patients (with fasting glucose > 140 mg/dL) and a normal healthy group. They found that the level of red cell reduced glutathione was significantly lower in type 1 diabetic patients but red cell glutathione peroxidase activity was significant‐ ly increased. The decrease of red cell glutathione may be due to its higher rate of consump‐ tion, increasing glutathione peroxidase activity or a reduction of pentose phosphate pathway, stimulated by insulin, resulting in lowered glutathione recycle (Likidlilid et al., 2007). In a recent study, reduced glutathione and vitamin E levels were decreased and ma‐ londialdehyde levels were higher in 20 type 1 diabetic children compared with healthy con‐ trols. After supplementation with vitamin E (600 mg/daily for three months), there was a significant decrease in malondialdehyde levels and significant increase in glutathione and vitamin E levels. This shows that vitamin E ameliorates oxidative stress in type 1 diabetic patients and improves antioxidant defense system (Gupta et al., 2011).

Other nonenzymatic antioxidants include α-lipoic acid, mixed carotenoids, coenzyme Q10, several bioflavonoids, antioxidant minerals (copper, zinc, manganese and selenium), and the cofactors (folic acid, vitamins B1, B2, B6, B12). β-carotene is a lipid soluble and chainbreaking antioxidant that effectively quenches singlet oxygen and inhibits lipid peroxida‐ tion. At low physiological oxygen pressures, it exhibits effective radical-trapping antioxidant behaviour (Frei, 1994). Coenzyme Q10 has been found to have a very important role in mitochondrial bioenergetics. It is an electron carrier-proton translocator in the respi‐ ratory chain and potent antioxidant which works by directly scavenging radicals or indirect‐ ly by regenerating vitamin E. In a study by Menke and colleagues (2008), plasma concentrations of coenzyme Q10 in 39 children with type 1 diabetes mellitus were higher than in healthy children. The findings suggest that elevated plasma concentration and the intracellular redox capacity of coenzyme Q10 in diabetic children may contribute to the body's self-protection during a state of enhanced oxidative stress (Menke et al., 2008). In an‐ other study, Salardi and colleagues (2004) determine whether serum hydroperoxides as oxi‐ dative markers and vitamin E and coenzyme Q10 as indexes of antioxidant capacity could be related to metabolic control in 75 unselected children, adolescents, and young adults with type 1 diabetes. Vitamin E and coenzyme Q10 were not significantly different from agematched control subjects. However, there were significant positive correlations between coenzyme Q10 and glycated hemoglobin, and vitamin E and glycated hemoglobin. It was al‐ so observed that diabetic patients with poor metabolic control and complications had elevat‐ ed vitamin E levels and coenzyme Q10 levels (Salardi et al., 2004).

Small molecules that have antioxidant capacity such as glutathione and uric acid are synthe‐ sized or produced within the body (Engler et al., 2003). A study by Maxwell et al. (1997) found significantly reduced total serum antioxidant status in 28 patients with type 1 diabe‐ tes mellitus as attributed by lower uric acid and vitamin C levels. Furthermore, multiple re‐ gression analysis showed that uric acid, vitamin E and vitamin C were the main contributors to serum total antioxidant activity.


*3.7. Relationship among Fruit Characteristics*

Correlation analysis was applied for investigating the interdependence of the guava characteristics (Figure 7). The results indicated that weight loss, decay, TSS, TSS/acid, total sugar, antioxidant, and rot had a negative relationship to firmness. This strong relationship revealed that greater guava had lesser antioxidant activity, which agrees with the resulted relationship for blackberries [41,42]. Strong positive correlations were found between firmness and acidity, V.C, and total chlorophyl. The marketable percentage was positively linked to firmness, acidity, V.C. and total chlorophyl; thus, these indicators could be used to forecast other results. Meanwhile, marketable percentage was negatively correlated with weight loss, decay, TSS, TSS/acid, total sugar, antioxidant, and rot, verifying that some physicochemical changes could cause lower acceptance by the consumers, and consequently create lesser marketability. PCA was utilized to discover the connection among the variables. Thus, PCA was utilized to discover the relationships among parameters in different treatments. PCA analysis revealed that the first principal component (PC1) and the second principal component (PC2) were 82.5 and 17.5%, respectively, with the accumulative variance contribution rate of 99% (>75–85%). PC1 was positively associated with the variables: VC, pectin, acidity, sugars, antioxidants, weight loss, firm, and decay area. PC2 was positively associated with the variables of TSS, phenolics, carotene, and chlorophyl. Furthermore, locations of the combined GA with natural extracts and the functional and freshness properties were close, showing the impact of GA-natural extracts on the quality of guava during the cold storage.

**Figure 7.** PCA analysis of the different treatments with different parameters.

### **4. Discussion**

Edible coatings have barrier features that decrease a fruits' surface permeability to oxygen and carbon dioxide, resulting in a change in internal gas composition that reduces oxidative metabolism and increases the fruit's shelf life [43]. We proposed that coating fruits with GA would result in significant differences, and increase the fruit shelf-life [20,44].

Water exchange between the interior and exterior atmospheres is believed to be the primary cause of fruit weight loss and decay percentage, resulting in a low marketable percentage during cold storage. With the advancement of cold storage, the growth in transpiration ratio, ethylene making, and the cellular interruption of fruits resulted in a rise in the physiological failure of weight and decay prevalence, and a reduction in saleable guavas [45–47].

Consequently, the use of GA can decrease the gases exchange among orange fruits and the environment by accumulating carbon dioxide in fruits with low O2-availability for respiration and, as a result, inhibit respiratory enzymes. Furthermore, GA coating can close openings in the peel. Furthermore, the coating can impede the fungi growth in a wide range of horticultural products [48]. This application improved membrane integrity, postponed fruit senescence, and reduced transpiration and respiration [49]. However, coating alters the atmosphere and inhibits the gaseous exchange of the fruit, which prevents ascorbic acid oxidation by limiting the entry of oxygen into the fruit's interior [50]. The lower TSS values in treated samples compared with the controls could be attributed to the conversion of organic acids to sugars via gluconeogenesis and the solubilization of cell wall ingredients by galactosidases and glucosidases found in guava fruit [51].

These findings are matched with those obtained by [25]; they indicate that guava fruits coated with 10% GA showed a significant reduction in weight loss (%) and a delay in the change in firmness, titratable acidity, soluble solid concentration color, maintaining the sensory quality during storage at room temperature, as compared with the uncoated control fruit of banana and papaya fruits, as well as Khaliq, Mohamed, Ali, Ding and Ghazali [23] in 'Choke Anan' mangoes, who reported that treated fruits with edible GA coatings had significantly higher firmness than uncoated fruits during cold storage. Moreover, in avocado fruit (Maluma), the application of GA at 10 or 15% + Moringa leaf extracts maintained higher firmness than other treatments [33]. A similar effect was found in mass loss, with fruit covered with the previously stated coatings showing minimal change. It may be argued that the prevention of moisture loss was the primary reason why coated fruit remained firmer than uncoated fruit. This theory is supported by [29], who found that moisture loss is not only associated with mass loss, but also fruit softening.

Cactus pear extracts and henna leaf extracts significantly decreased weight loss, compared with the control, during cold storage in both seasons. The antimicrobial and antifungal properties of henna leaf extract, as well as its antioxidant activity, contribute to its promising effect in delaying fruit weight loss and decay percentage. In addition, prickly pear has antimicrobial and antioxidant properties.

Lawsone makes up about 0.5 to 1.5 percent of the ingredients in henna. The main constituent responsible for the plant's dyeing properties is lawsone (2-dihydroxynaphthoquinone). Henna, on the other hand, contains mannitol, tannic acid, mucilage, and gallic acid. These substances are present in henna as a mixture. The antimicrobial activity could be attributed to many free hydroxyls that can combine with carbohydrates and proteins in the bacterial cell wall. They may become entangled with enzyme sites, proformance them inactive [52]. When compared with alcoholic and oily extracts, water extracts had no antibacterial activity. This could be due to a lack of solvent properties, which are key in antibacterial effectiveness.

Phenolic compounds are antioxidants that act as protective mechanisms in fruit. TPC content has a defense mechanism against plant pathogen invasion and plays an important role in plant resistance [2,53]. Furthermore, trapping the lipid alkoxyl radicals, antioxidants and phenols could significantly reduce reactive oxygen species (ROS) and prevent lipid peroxidation in plant tissue [2,54,55]. Maintaining TPC and increasing TAA could be attributed to postharvest treatments' ability to scavenge excess ROS and, as a result, reduce oxidative damage to the fruits [23,56,57].

Hence, the use of GA can reduce gas exchange among orange fruits and the environment by accumulating carbon dioxide in the fruits, resulting in low availability of O2 for respiration and, as a result, the reticence of respiratory enzymes. Furthermore, GA-coating can plug openings in the peel. Furthermore, a coating can prevent the growth of fungi in a variety of horticultural products [48].

### **5. Conclusions**

Overall, our findings indicated that both postharvest applications "cactus pear stem (10%), moringa (10%), and henna leaf (3%) extracts incorporated with gum Arabic (10%)" and their combinations had a positive impact on the quality characteristics of 'Maamoura' guavas during cold storage. The combined treatments of 10% GA + 10% moringa leaf extract were the most effective coating for fresh guava after long periods of cold storage. These applications significantly reduced weight loss, decay and *Rhizopus* rot infection (%), while also increasing marketable percentage, and delaying fruit softening during cold storage. Furthermore, these applications delayed color development by significantly retaining total chlorophyll content, maintaining fruit content in vitamin C and acidity, and slowing the accumulation of fruit contents in TSS and TSS/acid ratios compared with untreated fruits during the cold-storage period. Finally, compared with the control, these

applications significantly increased the shelf-life period at ambient conditions after the end of the cold-storage period.

**Author Contributions:** Conceptualization, M.F.M.A., M.S.G., A.M.E.B. and L.A.A.; methodology, M.F., A.R.M., M.H.M., H.M.A.E.G. and A.M.R.A.A.; software, H.M.A.E.G. and K.H.A.H.; validation, S.F.E.-G., M.S.G., A.M.E.B. and M.H.M. formal analysis, S.F.E.-G.; investigation, M.H.M. and H.M.A.E.G.; data curation, K.H.A.H.; writing—original draft preparation, S.F.E.-G., M.S.G., A.M.E.B., M.F.M.A. and K.H.A.H.; writing—review and editing, S.F.E.-G., M.S.G., A.M.E.B., M.F.M.A., M.F., A.R.M., A.M.R.A.A. and H.A.S.A.; funding acquisition, M.F.M.A., M.A.A., D.M.H. and L.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors extend their appreciation to King Saud University for supporting this work. Researchers Supporting Project under project number (RSP-2021/406), King Saud University, Riyadh, Saudi Arabia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Relevant data applicable to this research are within the paper.

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


**Abstract**

According to Grand View Research in polyglycerol market size, demand for diglycerol is expected to grow by 50% from 2012 to 2022 due to its extensive use in various industries, thus validating the importance and value addition of producing diglycerol. Due to the volatility of refined glycerine market price and increasing demand of diglycerol, research has been conducted to upgrade glycerol via various processes. Etherification is a single-step process of catalytic conversion of glycerol into polyglycerols, involving the condensation of two glycerol molecules to form the simplest oligomer which is diglycerol with linear, branched, or cyclic isomers. Thus, this chapter will discuss on the methods of synthesizing diglycerol followed by the type of catalyst to be used. These include homogenous and heterogenous catalyst with their subdivision of acid and base type, respectively. Besides, this chapter does include on the method for the etherification process where it highlighted the advantage of advance technology microwave irradiation over conventional heating.

**Keywords:** diglycerol, etherification, microwave, yellow glycerine, industrial

### **1. Introduction**

Glycerine, also called glycerol (propane-1,2,3-triol, C3H8O3), occurs as the backbone in triglycerides which are the main constituents of all vegetable and animal fats and oils. It is colorless and odorless, has sweet taste, and is very viscous and hygroscopic [1, 2]. Glycerol is a key ingredient in personal care product due to its property of acting as humectants, solvents, moisturizer, as well as an additive to make coatings. Glycerol is currently produced from biodiesel transesterification, saponification, and hydrolysis processes in fatty acid and soap production [3]. Among these, biodiesel transesterification is the biggest crude glycerol generation sector, contributing up to 67% of the total crude glycerol production, followed by high pressure splitting (17%) and soap production (<10%) [4].

Due to the growing biodiesel demand as substitution to petroleum-based diesel, the production of crude glycerol as by-product from this process is also increased. For every 1 ton of biodiesel produced, approximately 100 kg of glycerol will be generated [5]. The crude glycerol generated contains many impurities such as

methanol, organic and inorganic salts, water, unreacted triglycerides, soap, fatty acid, etc. Hence, for large-scale biodiesel manufacturers, the plants are normally equipped with crude glycerol refining facilities to produce glycerol with purities up to 95.5 and 99% [3].

etherification of glycerol under controlled conditions is required [7]. The etherification method has potential to replace the conventional epichlorohydrin route of producing polyglycerols which is relatively complex and involves in the production of toxic intermediate. Polyglycerols can be produced from different raw materials such as crude glycerine, yellow glycerine, as well as pure glycerine [7–9]. Hence, the economical production of glycerol derivatives is directly related with the

*Glycerol Conversion to Diglycerol via Etherification under Microwave Irradiation*

With regard to the heating method, microwave irradiation has been increasingly popular as a heating method for organic reaction. It had been proven to be more effective than the conventional water bath heating [10]. Various benefits of microwave irradiation included internal rapid heating that reduced the reaction time which compensates its higher power consumption. Besides, efficient and uniform heating that enables good temperature control can be achieved via microwave

On small scale, pure diglycerol is produced via direct synthesis methods in which diallyl ether is used as a primary reactant [1]. Diallyl ether is accessible by the reaction between allyl chloride and allyl alcohol in inert solvents under hydrogen chloride release. Direct hydroxylation of this product can be performed with peroxyformic acid, CH2O3, or permanganate at 40°C under safety precautions for 4.5 hours. However, several additional steps are needed for neutralization, filtration, derivatization, and fractional distillation which are required for the isolation of diglycerol and triglycerol. The isolation of diglycerol can be done using neutralization with barium hydroxide solution, centrifugation to separate the solid, digestion of the product in absolute ethanol, and fractional distillation under reduced

The thermal reaction for glycerol oligomerization is conducted at a certain temperature under inert protecting reactor condition [1]. For selective reaction, pure glycerol up to 99% purity should be used. Commonly, before the use of the oligomeric products for further reactions, a distillation is needed to remove

unconverted glycerol. Reaction temperature, basicity, and organic impurities would affect the glycerol oligomerization. For pure thermal conversion of glycerol without the addition of catalyst, temperature is normally set above 200°C. However, at reaction temperature higher than 290°C, side products with strong smelling are formed. At 180°C (low temperature) with the addition of alkali catalyst, the formation of diglycerol from glycerol is observed but at low conversion of glycerol.

The epichlorohydrin method of producing polyglycerol is commonly applied. It involves basic hydrolysis by sodium hydroxide, led to the formation of intermediate product, namely, glycidol and glycerol, and it will react with unconverted epichlorohydrin or glycerol to diglycerol [6]. Further separation and purification of

quality of glycerine feedstocks and prices.

*DOI: http://dx.doi.org/10.5772/intechopen.90513*

**2.1 Existing diglycerol synthesis method**

heating.

pressure.

**119**

**2. Glycerine conversion**

*2.1.1 Laboratory-scale routes*

*2.1.2 Thermal conversion of glycerol*

*2.1.3 Epichlorohydrin method*

Generally, there are three types of glycerol, namely, crude glycerine, technical grade glycerine, and refined glycerine. The process of refining glycerine is a complex process which involves distillation, bleaching, acidification, and several intermediate steps. In the distillation step, refined glycerine is obtained from the side draw of the distillation column and then subsequently bleached and deodorized to achieve the refined grade. Technical grade glycerine, also called yellow glycerine, is obtained from the top of the column with some impurities such as water, fatty acids, soap, etc. [2]. **Figure** 1 shows the general process flow of glycerine refining.

According to MarketWatch 2018, the global glycerine market is expected to reach approximately 6200 metric tons by 2024 from 3550 metric tons in 2016, resulted from the growth in bio-renewable chemicals, biodiesel production, and wide range of applications. However, with regard to the market price, technical grade glycerine is cheaper than the refined glycerine which are approximately RM4400 and RM5400 per metric ton, respectively, as reported by Oleoline (2017).

Furthermore, glycerol can be used as a starting material for the synthesis of value-added chemicals via catalytic conversion such as hydrogenolysis to propanediol and ethylene glycol, dehydration to acrolein, fermentation to propane-1,3-diol, thermal reduction into syngas, etherification to fuel oxygenates, conversion into glycerol carbonate, and synthesis of epichlorohydrin [6]. Among these, the etherification of glycerol into polyglycerols particularly diglycerol (DG) and triglycerol (TG) is gaining more interest in the recent research due to their possibility in controlling the hydrophilic–lipophilic balance (HLB) which is highly important as additive to food and pharmaceutical industries, lubricants, stabilizers, dispersants, plasticizers, etc. [6]. According to Grand View Research in polyglycerol market size, the demand for diglycerol and triglycerol is expected to grow by 50% from 2012 to 2022. The global diglycerol demand is expected to grow at a CAGR of 5.3% due its extensive use in various industries, thus validating the importance and value addition of producing diglycerol.

Due to the volatility of refined glycerine market price and increasing demand of polyglycerol especially the shortest chain oligomer which is diglycerol, research has been conducted to upgrade glycerol via various processes such as pyrolysis, epichlorohydrin, and catalytic etherification to produce polyglycerols. Etherification is a single-step process of catalytic conversion of glycerol into polyglycerols, involving the condensation of two glycerol molecules to form the simplest oligomer which is diglycerol with linear, branched, or cyclic isomers. Further reaction yields tri-, tetra-, and higher oligomers. Short-chain oligomers are preferred. Therefore, the

**Figure 1.** *General process flow of glycerine refining.*

#### *Glycerol Conversion to Diglycerol via Etherification under Microwave Irradiation DOI: http://dx.doi.org/10.5772/intechopen.90513*

etherification of glycerol under controlled conditions is required [7]. The etherification method has potential to replace the conventional epichlorohydrin route of producing polyglycerols which is relatively complex and involves in the production of toxic intermediate. Polyglycerols can be produced from different raw materials such as crude glycerine, yellow glycerine, as well as pure glycerine [7–9]. Hence, the economical production of glycerol derivatives is directly related with the quality of glycerine feedstocks and prices.

With regard to the heating method, microwave irradiation has been increasingly popular as a heating method for organic reaction. It had been proven to be more effective than the conventional water bath heating [10]. Various benefits of microwave irradiation included internal rapid heating that reduced the reaction time which compensates its higher power consumption. Besides, efficient and uniform heating that enables good temperature control can be achieved via microwave heating.


**8. Conclusions**

BoNT-A injections are widely used in functional urology disorders, and therapeutic benefits have been noticed across a wide range of lower urinary tract diseases. However, approval for indication has been obtained only for management of idiopathic OAB and NDO owing to SCI or multiple sclerosis. The United States and some other countries also approved use for the detrusor hyperactivity with impaired contractile function. Moreover, for IC/BPS patients' refractory to medical treatment, BoNT-A injections are considered the standard of treatment according to different guidelines. The pathophysiology of BoNT-A injection to different functional disorders is increasingly revealed in various clinical trials, and with promising therapeutic results of novel BoNT-A applications, including DV, BPH, and chronic prostatitis. However, not all patients of functional urology disorders refractory to conventional therapy can benefit from BoNT-A treatment, possibly because the underlying pathophysiology of these diseases has not been entirely clarified.

**Funding:** This research was funded by Ministry of Science and Technology (107-2314-B-567-002 MY2 and 108-2628-B-567-001).

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


**Abstract**

The purpose of this study is to clarify the effects of the smelling "Zuko", incense rubbing into hands and putting the hands for the human brain. From our previous studies on the smelling an incense odor with putting the hands together, the activities of "Zuko" incense are also considered to be promoted as the imitation of habitual behaviors by mirror neurons and the default mode network in our brain. In this experiment, the brain activation was measured in 10 healthy adult volunteers who did or did not have a habit of putting their hands together and magnetoencephalography (MEG) data were recorded while the participants smelled "Zuko" incense and putting their hands together. The peak response of MEG P300m for the "auditory odd-ball paradigm" was also measured for a rare auditory pulse stimulation and was more enforced by the smelling "Zuko" incense. We used alpha-amylase value as an index of the stress state measured in the state before and after smelling "Zuko" and MEG experiments. From these results it can be considered that smelling "Zuko" promote the excitation of the higher activities to human brain and make changing the specific brain areas such as OFC, F5 and V1.

**Keywords:** "Zuko" rubbing into the hands, OFC, P300m, alpha-amylase, MEG, spatiotemporal dipole fit, time-varying analysis