**6. Pseudo-optimal choice of the preferred lay-ups**

In the final stage of investigation, the optimizing structure made of the preferred lay-ups "candidates" has been loaded simultaneously by the systems of three forces: bending by the

**Figure 9.** Dependencies of maximum deflections and torsion angles of the loaded beams.

values of effective elastic moduli for the laminate with given lay-up. Note that according to the homogenization hypothesis this laminate is considered as the solid body with

In the next stage of investigation, the angular dependencies of the effective moduli were investigated. In order to avoid the significant reduction of the structural rigidity in some directions, these dependencies should not have sharp drops, corresponding to these directions. Some examples of such angular dependencies are shown in **Figure 8**. These dependencies have been used for the preliminary selections of the "candidates" lay-ups, which have been further used

The mechanical testing of the structure with different lay-ups and ply stacking angles has been implemented using FE model (see **Figures 1**–**3**) by using three abovementioned types of static bending and torsion loading. These loads were applied separately; for each load

deflections max(*vb*), max(*wb*) and torsion angles *θ* on the free face of the beam tip were calculated. At the solving of these subtasks a linear parametric solver was used. Results of these testing were used for the refinement of the "candidates," that is, select lay-ups that provide the minimum strain energy, bending and torsion deformations. Our study demonstrated a strong coupling of total strain energy of deformed structure with the values of maximum beam deflection (bending load cases) and maximum torsion angles (twisting load case). Moreover, two very interesting and important facts were established. First, we found that sensitivity of both maximum and averaged von Mises stress to the structural symmetry of orthotropic material is noticeably less comparing to the total elastic strain energy. Thereby we do not give here any von Mises dependencies. Second result is the practically independent response of the structure on the twisting load of the lay-ups II, III and IV. The reason for this is the practical identity of the shear modulus of these lay-up schemes at the same

The calculated dependencies of maximum deflections and torsion angles of the loaded beams on the lay-up parameters are present in **Figure 9**. These results together with the angular dependencies of elastic moduli that are partially presented in **Figure 8** allowed to select eight "candidates" for further optimization. These candidates had to have acceptable values for the

In the final stage of investigation, the optimizing structure made of the preferred lay-ups "candidates" has been loaded simultaneously by the systems of three forces: bending by the

*tot*, the maximum and averaged von Mises stress, two end

to test them in mechanical testing of the optimized slender structure.

**5. Mechanical testing of structure with different laminate lay-ups**

uniform structure.

46 Optimum Composite Structures

scenario the total strain energy *Eel*

lamina angular orientation (see **Figure 7b**–**d**).

total strain energy and rigidity under all loading scenarios.

**6. Pseudo-optimal choice of the preferred lay-ups**

distributed forces oriented along y and z axis and twisting torque applied to the external surface and given by Eqs. (1) and (2). The studied responses included total strain energy, the maximum bending deflections and objective, which was accepted in the normalized dimensionless form.

$$Obj = \mathcal{O}/\mathfrak{G} + \max\{\upsilon\_i\} + \max\{\upsilon\_i\}/1.5\tag{5}$$

Where, *θ* is the torsion angle and *vb*

function determined by Eq. (5).

*Eei*

; *φin* = 150

tion procedures, we used such "candidates": (I *φin* = 300

; *φin* = 150

); (V *ψ* = 450

imposed.

(V *ψ* = 300

the preferred lay-ups).

, *wb*

tively. Naturally, definition of such objective function and weights for its components has a significant element of arbitrariness. Therefore, the total strain energy, the beam deflections and torsion angle were also monitored during optimization process along with the objective

For the definiteness of the optimization problem statement the following constraints were

At the optimization workflow, the forward structural mechanics problem has been called by the built-in gradient-free Nelder-Mead optimizer. In order to start the iterative optimiza-

**Figure 11.** Comparison among the best lay-ups of the optimized structure (stems with the filled symbols correspond to

; *φin* = 150

*tot* ≤ 160*J* ; *θ* ≤ 4.50 ;

); (V *ψ* = 600

are the beam tip deflections along the *y* and *z*, respec-

http://dx.doi.org/10.5772/intechopen.76566

Optimization of Lay-Up Stacking for a Loaded-Carrying Slender Composite Beam

max(*vb*) ≤ 0.46*m* ; max(*wb*) ≤ 0.86*m* (6)

); (II *φin* = 300

) and (V *ψ* = 750

); (III *φin* = 300

; *φin* = 300

); (IV *φin* = 300

), which had

);

49

**Figure 10.** Evolution of lay-up angle, objective and mechanical properties of structure during optimization for two "candidate" lay-ups: (I *φ in* = 30 0) and (V *ψ in* = 30 0; *φ in* = 15 0).

Where, *θ* is the torsion angle and *vb* , *wb* are the beam tip deflections along the *y* and *z*, respectively. Naturally, definition of such objective function and weights for its components has a significant element of arbitrariness. Therefore, the total strain energy, the beam deflections and torsion angle were also monitored during optimization process along with the objective function determined by Eq. (5).

For the definiteness of the optimization problem statement the following constraints were imposed.

 *Eei tot* ≤ 160*J* ; *θ* ≤ 4.50 ; max(*vb*) ≤ 0.46*m* ; max(*wb*) ≤ 0.86*m* (6)

At the optimization workflow, the forward structural mechanics problem has been called by the built-in gradient-free Nelder-Mead optimizer. In order to start the iterative optimization procedures, we used such "candidates": (I *φin* = 300 ); (II *φin* = 300 ); (III *φin* = 300 ); (IV *φin* = 300 ); (V *ψ* = 300 ; *φin* = 150 ); (V *ψ* = 450 ; *φin* = 150 ); (V *ψ* = 600 ; *φin* = 150 ) and (V *ψ* = 750 ; *φin* = 300 ), which had

**Figure 11.** Comparison among the best lay-ups of the optimized structure (stems with the filled symbols correspond to the preferred lay-ups).

**Figure 10.** Evolution of lay-up angle, objective and mechanical properties of structure during optimization for two

"candidate" lay-ups: (I *φ in* = 30 0) and (V *ψ in* = 30 0; *φ in* = 15 0).

48 Optimum Composite Structures

better results at the previous stage of the study (see **Figure 9**), where Roman numeral indicates the type of lay-up (see **Figure 4**) and the value of angle is its start value.

of quasi-optimal structure. The short discussion of the obtained results confirms necessity of multiobjective approach to the studied optimization problem, taking into account many requirements and constraints that allows to make the final choice of the best lay-up parameters.

This work is implemented in the framework of the state mission for the South Scientific Center of Russian Academy of Science, project No. AAAA-A16-116012610052-3 and it was supported by the Russian Foundation for the Basic Research (Grant 15-08-99849 and Russian-Taiwanese

, Natalia Snezhina2

3 National Kaohsiung University of Science and Technology, Kaohsiung City, Taiwan R.O.C.

[1] Baker A, Dutton S, Kelly D. Composite Materials for Aircraft Structures. 2nd ed. Reston:

[2] Ostergaard MG, Ibbotson AR, Le Roux O, Prior AM. Virtual testing of aircraft structures.

[3] Taylor JE. A formulation for optimal structural design with optimal materials. In: Rozvany GIN and Olhoff N, editors. Topology Optimization of Structures and Composite Continua. Dordrecht/Boston: Kluver Academic Publishers; 2000. pp. 49-55.

[4] Rao JS. Advances in aero structures. In: Proceedings of 12th International Conference on

[5] Gillet A, Francescato P, Saffre P. Single- and multi-objective optimization of composite structures: The influence of design variables. Journal of Composite Materials.

[6] Optimization of Composite: Recent Advances and Application [Internet]. 2011. Available

CEAS Aeronautical Journal. 2011;**1**:83-103. DOI: 10.1007/s13272-011-0004-x

Vibration Problems (ICOVP-2015); December 14-17, 2015; Guwahati. p. 20

from: www.altairproductdesign.com [Accessed: February 01, 2018]

and WU Jiing-Kae3

Optimization of Lay-Up Stacking for a Loaded-Carrying Slender Composite Beam

http://dx.doi.org/10.5772/intechopen.76566

51

**Acknowledgements**

project 16-58-52013 MNT\_a).

\*, Igor Zhilyaev1

\*Address all correspondence to<EMAIL_ADDRESS>1 South Center of Russian Academy, Rostov on Don, Russia

2 Don State Technical University, Rostov on Don, Russia

AIAA Inc.; 2004. p. 597. ISBN 1-56347-540-5

2010;**44**(4):457-480. DOI: 10.1177/0021998309344931

**Author details**

Sergey Shevtsov1

**References**

ISBN 0-7923-6806-1

**Figure 10** illustrates two examples of objective and other responses evolution during optimization process. These plots demonstrate very fast convergence to the quasi-optimal solutions for both optimized lay-up stacking. These solutions are not global optimum because they characterized by the different values of all responses.

The final features of all optimized "candidate" structures are present in **Figure 11**. All optimized lay-ups were ranked according to calculated responses. The optimized lay-ups (I *φin* = 300 ) and (V *ψ* = 300 ; *φin* = 150 ) provide minimal values of objective, but (II *φin* = 300 ) provides the minimal total strain energy. Lay-up (V *ψ* = 450 ; *φin* = 150 ) provides the greatest torsional stiffness, but its flexural rigidities are downscale. These considerations substantiate obligatoriness of multiobjective optimization at the design of load-carrying multilayered composite structures with orthotropic symmetry of the materials. The final decision can be made taking into account some constraints and requirements, for example, complexity of manufacturing, weight of ready structure, the natural vibration modes and eigenfrequencies and importance of a particular rigidity for the operability of the structure. An additional study of the strength of the composite layers according to the Tsai-Wu criterion [25] should be carried out at critical loads. Meanwhile, the used approach requires many tedious calculations, but allows us to obtain the visual substantial representations about chosen composite lay-ups for optimizing structures, and can be expanded to the composite structures with arbitrary number of arbitrarily oriented unidirectional layers.


As well as behavioural responses and reaction times, the length of the spider (mm), height (cm) and length (cm) of the capture spiral were measured using a standard 30 cm ruler. The capture spiral area was calculated using the ellipse-hub equation (vertical diameter/2 × horizontal diameter/2 × π—(hub diameter/2)2) [34]. The temperature ( ◦C) and humidity (%rH) were measured using an ETI 810-190 Pocket-Sized Thermo Hygrometer (Electronical Temperature Instruments Ltd., Worthing, U.K.)).

**Table 1.** Ethogram of anti-predatory and predatory behaviours in *Metellina segmentata*. The ethogram is split into three categories, attack, escape, and no response.

#### *2.4. Data Analysis*

For the 2020 method, a total of 50 spiders were observed during the initial data collection, and 38 were subsequently used in the data analysis. For the 2021 method, 46 spiders were observed during the initial data collection, and 40 were used in the subsequent data analysis. Spiders were removed due to insufficient data, primarily caused by not returning between stimuli. A Fisher's Exact test for contingency tables [35] was used to compare frequency distribution across the different stimuli and across the years, as well as between the order in which the stimuli were presented, as a Chi-square contingency test could not be used due to many expected values being below 5. We developed linear mixed models (using the *lmer()* function from the lme4 package) in R [36] to test whether the type of behavioural response (using only the data where the spider responded either by attacking or escaping) varied with spider size and capture spiral area, where the latter were response variables and behavioural response a fixed variable. Site and year were included as random variables.

For all models, we log-transformed the response variables to achieve normality of the residuals and *p* values were estimated from the Type II Wald F tests with Kenward–Roger degrees of freedom. We used R [37] for all statistical tests with a significance level of 0.05.

#### **3. Results**

#### *3.1. Behavioural Responses*

The produced ethogram shows that our spiders displayed a wide range of responses to the different stimuli, which we could categorise into either attack, escape, or no response behaviours (Table 1). The attack behaviours were the most complex of the three categories and generally involved moving towards the stimuli and in some instances physically interacting with the tuning fork or wire. Interestingly, unlike for escape or no response behaviours, the spiders reacted to the wire with different attack behaviours than they did to the tuning forks. The most common reaction to the tuning fork was an initial aggressive movement towards the tuning fork, but either pausing or dropping from the web before it came into contact with it. The most frequent escape response was to rapidly drop off the web (Table 1). The small proportion of spiders that did not respond mainly showed no visible reaction, although a few flinched slightly (Table 1).

In order to better quantify spiders' reactions, we analysed the behaviours in the larger categories, attack, escape, or no response. We found a significant difference in the responses to the three stimuli for both 2020 (Fisher's Exact Test, *p* < 0.0001) and 2021 (Fisher's Exact Test, *p* < 0.0001), with spiders predominantly escaping in response to the 256 Hz tuning fork, almost exclusively attacking in response to the 440 Hz and either attacking or escaping in response to the wire (Figure 1). The number of spiders not responding remained low for all stimuli, with the highest proportion of 16% found for the wire in 2020 (Table 1).

**Figure 1.** Behavioural responses of *Metellina segmentata*, showing the number of attack, escape, and no response behaviours seen for each stimulus. Split into 2020 method (**A**–**C**) and 2021 method (**D**–**F**).

The behavioural responses from the 2020 method and 2021 method were consistent with each other (Figure 1). There were no significant differences in the frequency distributions of responses between 2020 and 2021 for the 256 Hz tuning fork (Fisher's Exact Test, *p* = 0.81), the 440 Hz tuning fork (Fisher's Exact Test, *p* = 0.51) or the wire (Fisher's Exact Test, *p* = 0.49).

#### *3.2. Order of Stimuli*

We found no clear influence of the order of stimuli (which was consistent in 2020 and randomised in 2021), as behavioural responses were similar independently of the order in which they were presented to the spider for both the 256 Hz (Figure 2A–C; Fisher's Exact Test, *p* = 0.584) and the 440 Hz tuning fork (Figure 2D–F; Fisher's Exact Test, *p* = 0.333). However, the behavioural responses to the wire showed less consistency (Figure 2G–I; Fisher's Exact Test, *p* = 0.033). When the wire used as the first stimulus, the primary response was to attack (Figure 2G), whilst when it was the second stimulus, the primary response was to escape (Figure 2H). These results are further confounded by almost equal attack and escape behavioural responses when it was the third stimulus (Figure 2I).

**Figure 2.** The influence of the order of the stimuli on the behavioural responses of *Metellina segmentata*. (**A**–**C**) 256 Hz tuning fork; (**D**–**F**) 440 Hz tuning fork; (**G**–**I**) length of wire.

#### *3.3. Effect of Size and Web Area*

In order to showcase some of the quantitative questions that can be answered using these methods, we compared behavioural responses (attack or escape only) between spiders of different sizes and with different sized webs for the full data-set, disregarding year and order of stimuli as they had no large impact on the behavioural response as demonstrated above. We found that statistically, the length of the spider did not differ between behavioural responses to 256 Hz (F = 2.41, df = 1, 67.5, *p* = 0.13), 440 Hz (F = 1.20, df = 1, 68.5, *p* = 0.28), or the wire (LMM: F = 0.003, df = 1, 63.8, *p* = 0.96). Similar results were obtained with web area, where we also found no differences in behavioural response to 256 Hz (F = 1.22, df = 1, 67.8, *p* = 0.27), 440 Hz (F = 0.47, df = 1, 69.1, *p* = 0.49) or the wire (F = 2.57, df = 1, 63.4, *p* = 0.11). Nonetheless, the average length of spiders and the average capture spiral area tended both to be larger in spiders that attacked (Figure 3). Whilst

for the wire, the size of the spider had no impact on the behavioural response observed, although spiders that escaped tended to have a larger capture spiral (Figure 3).

**Figure 3.** Comparison of the length of the spider (mm) and the area of the capture spiral (cm2) against the behavioural responses observed in *Metellina segmentata.* (**A**,**D**) 256 Hz tuning fork; (**B**,**E**) 440 Hz tuning fork; (**C**,**F**) length of wire.

#### *3.4. Reaction Times*

The stimulus type (tuning forks or wire) and the behavioural response (attack or escape) both had a significant impact on the reaction time of the spider (Behaviour: F = 22.8, df = 1, 202.0, *p* < 0.0001; Stimulus: F = 4.09, df = 2, 200.3, *p* = 0.018) and the interaction between them was also significant (F = 9.47, df = 2, 201.5, *p* = 0.0001). Interestingly, whilst spiders had faster reactions when escaping the 256 Hz tuning fork, they reacted faster when they attacked the 440 Hz tuning (Figure 4). In response to the wire, the difference was minimal, although there was a slight tendency for them to react faster when escaping (Figure 4).

**Figure 4.** Reaction times (ms) of the attack and escape behavioural responses of *Metellina segmentata* for the three stimuli. (**A**) 256 Hz tuning fork; (**B**) 440 Hz tuning fork; (**C**) Metal wire.

#### **4. Discussion**

Our results confirmed that tuning forks can be utilised to study the behavioural responses of orb web spiders. The behaviours demonstrated by *M. segmentata* are in accordance with the secondary defences described by Cloudsley-Thompson [10] and Tolbert [11] in that we observed both rebuff (attacking the stimuli) and flight (running away from the stimuli). The most common escape behaviour in our study was dropping from the web, which was also shown sometimes following attack behaviour. Dropping from the web on a safety thread to enable an easy return to the web after the danger has passed has been described as common in both araneid and tetragnathid spiders [10,38]. In the ethogram developed based on our study, we broadly categorised the behaviours as attack, escape, and no response, with the first group showing the largest number of distinct behaviours. Behaviours in this group also showed a combination of attacking and escaping, with some spiders moving towards the location of the tuning fork, and then escaping by dropping from the web. It should be stated that the spiders observed within this experiment never exhibited the common anti-predatory response of web shaking. Defined by Willey et al. [39] as "violent, large amplitude, movements of the web surface", this behaviour has been observed in *Cyrtophora* [40] and *Mecynogea* [39]. Tolbert [11] called it web flexing, and observed the "Spider and web [swinging] back and forth parallel to the ground ... " in *Argiope* and *Araneus* spp. This behaviour has been observed in the tetragnathid *Azilia vachoni* (Caporiacco, 1954) [38], but it is possible that the Meteine group of tetragnathids do not show this anti-predator behaviour.

The replication of results from three different methods, over two years, demonstrates the feasibility of obtaining reliable behavioural data using tuning forks, and the ease at which they can be used in the field confirm their suitability. The repetition in behavioural responses for the two tuning forks, regardless of the order the stimulus was presented, further demonstrates the reliability and effectiveness of the method. Directly stimulating

spiders mechanically with a wire was, while still generating a clearly defined escape or attack behaviour, less reliable. Additionally, the behavioural responses to this stimulus appeared to be dependent on the order of its use; the change from attack to escape when it is the second and third stimuli could be due to the spider becoming sensitised to the prior stimuli. For *M. segmentata*, the 440 Hz tuning fork is the ideal frequency to simulate predators, as the behavioural responses to this frequency are principally attacking (90% of responses for both years combined). The 256 Hz tuning fork largely produces escape behaviours (58% of responses across the two years), although there was more variation with this tuning fork (36% showed attack behaviours). This could mean that a lower frequency is required to produce the ideal simulation of a predator as larger, and hence potentially more dangerous, insects tend to vibrate their wings between 100 Hz and 250 Hz [33]. Alternatively, this inter-individual variation could be attributed to behavioural syndromes, defined by Sih et al. [41] as "a suite of correlated behaviors reflecting between individual consistency in behavior across multiple (two or more) situations". Behavioural syndromes can explain the tendency of some spiders to always attack or always escape from certain stimuli. For example, there are aggression syndromes, where across a range of situations, some individuals are more aggressive, whilst others are less aggressive [41]. The ease of using the tuning fork method in the field mean it could potentially be used to study behavioural syndromes in spiders in the wild.

The size of the spider (here measured as total length) in our study did not significantly affect whether they responded to the stimulation with attack or escape behaviours, although there was a weak trend across the two tuning forks for the attacking spiders being slightly larger than those that escaped. Similarly, the size of the web (here measured as capture spiral area) did not differ between those spiders that attacked or escaped. However, again there was a weak trend for spiders showing attack behaviour in response to the tuning forks to have larger webs, although an opposite trend was found in response to the wire. Our findings are supported by a previous study relating female aggressiveness towards males and prey in the araneid spider *Larinioides sclopetarius* (Clerck, 1757), which found that aggressiveness was largely independent of female size [42].

Our findings further demonstrate how tuning forks can be used to collect quantitative data such as the spider's reaction time to the presented stimuli. Reaction time has been a widely used measure for quantifying foraging behaviour [13,14], and it has also been used to estimate motivation or preparedness of the spider to repair webs under different wind loadings [16]. In our study, spiders showed the fastest reaction time to the wire, and the slowest responses to the 256 Hz tuning fork. However, interestingly the 256 Hz tuning fork elicited a number of spiders to react with escape behaviours before the tuning fork came into contact with the web, thereby producing a negative reaction time. This occurred across the 2020 and 2021 methodologies. This suggests that *Metellina* spiders are able to detect air-borne movements of predator species similar to reactions of airborne vibrations from prey demonstrated in araneid orb spiders [43]. This phenomenon can be explained by trichobothria, which are cuticular filiform hairs that respond to air movements and can be found on the legs and pedipalps of spiders [44,45].

The effectiveness of using tuning forks still needs to be investigated in more detail using more orb spider species and a wider range of frequencies in order to confirm our findings that show that orb web spiders tend to respond to high frequencies with prey capture behaviour, and to low frequencies with anti-predatory behaviours. Given that Nakata and Mori [29] found that both the small-to-medium-sized araneid *Cyclosa argenteoalba* (Bösenberg and Strand, 1906) (similar in size to *M. segmentata*) and the larger araneid *Eriophora sagana* (Tanikawa, 2000) show anti-predator responses to a 440 Hz tuning fork, it is clear that there are family-, environmental- or species-specific factors at play. However, even if the application of the methods in this study should turn out to be limited to Tetragnathid spiders in the Meteine group, the use of tuning forks could still be of potential use in studying the behaviour of Meteine cave spiders [46]. Studying the behaviour of cave animals in general is difficult due to the impediments caused by high sensitivity to

disturbances, low population densities and the practical difficulties of observers spending a long time in caves [47]. So, developing easy and quick methods, such as using tuning forks, as demonstrated in this study, is essential. Anti-predatory behaviours are especially interesting to study in caves, as their low nutrient availability results in few species, and hence few predators, which again should reduce any anti-predatory responses [48]. Thus, comparing responses to the 256 Hz tuning fork at different distances into the cave and between spiders with different degrees of behavioural adaptations to the subterranean habitat, such as *Meta menardi* (Latreille, 1804), which modifies its orb web geometry [49], and *Metellina merianae* (Scopoli, 1763), which does not [50], would shed light on the interplay between evolution and phenotypic plasticity (flexibility in web-building behaviour in response to environmental factors) in shaping the behaviours of cave animals.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/insects13040370/s1, Video S1: *Metellina segmentata* showing attack behaviour in response to a 256 Hz tuning fork. Video S2: *Metellina segmentata* showing escape behaviour in response to a 256 Hz tuning fork. Video S3: *Metellina segmentata* showing attack behaviour in response to a 440 Hz tuning fork. Video S4: *Metellina segmentata* showing attack behaviour in response to direct stimulation by a metal wire.

**Author Contributions:** Conceptualization M.S.D. and T.H.; methodology M.S.D. and T.H.; data collection M.S.D.; original draft preparation M.S.D.; writing—review and editing T.H.; supervision T.H. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** This study did not require any ethical approval.

**Data Availability Statement:** Link to Figshare will be made available after manuscript acceptance (https://figshare.com/articles/dataset/Response\_of\_an\_orb\_spider\_to\_tuning\_forks/19165460).

**Acknowledgments:** The authors would like to thank Newport City Council, Caerphilly City Council and Woodland Trust for providing permission to research on the sites. MSD would like to thank Raymond Davies for assistance in the field.

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

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**3. Chemodiversity explodes with the emergence of life**

### **3.1. "Cool" carbon chemistry**

amino acids and their non-enzymatic condensation into dipeptides or small oligopeptides within the lipid bilayer. In addition, vectorial properties in phospholipidic vesicles are thought to influence the inner pH (making it lower than outer pH) in small vesicles, due to differences

**Figure 3.** A phospholipid vesicle, with a double-layered membrane. Each layer includes a hydrophilic head (dark knobs) and a hydrophobic tail made up of long lipid chains. The aqueous environment inside the vesicle isolates en‐

The total synthesis of a simple cell is possibly the ultimate challenge in synthetic biology. It is experimentally possible to entrap nucleic acids inside self - forming phospholipid vesicles, acting as reaction vessels for e.g. polymerase chain reaction, enzymatic RNA replication and even protein synthesis, as demonstrated by Oberholzer and collaborators (reviewed in [19]). The molecular pump scenario [20], in which peptides were initially synthesized through a molecular engine could also have taken place inside such structures. In nature, the inclusion of clay microparticles acting as catalysts is highly probable. Thus, phospholipid vesicles can be regarded as useful experimental models of proto-cells [21], but many questions still need

*Protobiology* uses a bottom up approach when designing complex and functional protocells

*Synthetic biology* uses a top-down approach to identify the minimal components of living cells that will qualify, in other words to rediscover the basic cell before it underwent the complexities of Darwinian evolution. Thus protocells are of exciting biotechnological interest in the quest

Prebiotic chemistry is certainly more complex than previously thought, and chemodiversity has undergone periods of expansion, especially through stochastic combinations during the

to optimize chemotherapies (cellular target recognition, kinetics of drug delivery).

trapped metabolites, biopolymers and catalysts, the whole structure acting as a reaction vessel.

addressing before considering them as early precursors of living cells.

so-called RNA world, and during the development of proto-cells.

from simple molecules and organic catalysts.

in the behavior of water molecules.

76 Biodiversity - The Dynamic Balance of the Planet

*2.3.3. Vesicles as proto-cells*

Our chemodiversity is usually classified as mineral or organic. Exploitable minerals are solidified in the earth crust or deposited as sediments or conglomerates - geochemical cycles connect the molten inner core of the planet with the biosphere through the displacement of continental plates and volcanic activity. Organic chemodiversity is concentrated in the biosphere, as the result of the present and past metabolic processes of marine and terrestrial organisms. Chemically, organic molecules are carbon atoms and scaffolds of connected carbon atoms, linked to hydrogen and covalently to heteroatoms e.g. oxygen, nitrogen, atoms but also to sulfur, phosphorus and halogens or metal complexes (*coordination chemistry*) to form functional groups. Larger organic molecules often adopt three-dimensional architectures that confer them with the exquisite specificity required for recognition and binding to a defined molecular target. Organic natural products are by far the largest contributors to marine and terrestrial chemodiversity, and also the most labile in the face of anthropic and natural influences that affect the stability of our ecosystems.

#### **3.2. "Cool" oxygen biochemistry**

The chemistry of our oceans has evolved considerably [22], especially with the gradual production of oxygen, which initially oxidized mantle rocks and dissolved reduced metals such as iron then tended to precipitate, as evidenced by red strata in ancient sediment beds. Free oxygen then started to rise in atmospheric concentrations, some of which was split by cosmic radiations to form the protective ozone layer. Respiration became possible and some life forms moved out of the oceans, and both aquatic and terrestrial species diversification started, and with it, organic chemodiversity. It should be emphasized that combustion occurs at oxygen levels only above 15% and organic matter would spontaneously burn at 25% concentrations - which explains that life is possible only if the present level (21%) is maintained around 20% and if genotoxic radiations can be efficiently filtered out. Oxygen is also toxic at the cellular level, if the production of (or exposure to) reactive oxygen species (ROS) or oxygen radicals cannot be counterbalanced by antioxidants or if the resulting damages cannot be repaired. When involved in regular intracellular signaling, ROS can be involved in the activation of defense metabolic pathways, and contribute to the enrichment of the metabolic repertoire of planktonic and benthic organisms.

#### **3.3. Biochemistry operates under very specific conditions**

With the exception of very few extremophiles that can live under elevated temperatures (above 80°C) and hyper acidic environments pH < 2), most microorganisms are not tolerant to conditions found outside the biosphere. The biosphere represents a very narrow region less than one thousand times thinner than the globe's diameter, and comparable conditions have not been found on any other planet so far. This does not mean that dormant bacterial spores could not withstand intersidereal journeys - lichens have successfully resisted prolonged exposures in outer space during space craft experimentations. High biodiversity environments are usually found in warm, stable environments where nutrient cycling is efficient (without accumulation of wastes) - this is at least true for tropical rainforests and shallow water coral reefs.

such changes in microbial life or on the putative role microbial associates had on the *reinven‐ tion* (understand: adaptive evolution) of new species. As Falkowski [25] puts it, animals and plants are merely new incarnations of ancient metabolic processes, but the ultimate key to biodiversity may be held by bacteria ferrying the set of core genes that are necessary for life

Marine Biodiversity and Chemodiversity — The Treasure Troves of the Future

http://dx.doi.org/10.5772/57394

79

Water is an extraordinary medium and, in many respects, mysterious. Its outstanding solvation properties are due to the capacity of the water dipoles (H-OH) to dissociate, reorientate and accommodate salts and polar compounds in a monophasic system [27]. Salts are essential to maintain osmotic balance and membrane polarization. Hydration is essential for the catalytic properties of enzymes. Semiotic (communication) substances are usually released as pheromones (intra-specific signaling) or allomones (against defense or predation). In addition, apolar (long chains or cyclic) molecules can be associated with mucus and dispersed. Some chemical cues are surface-adsorbed on dead substratum and serve as transgeneration signaling, e.g. for the settlement of larvae near adult colonies. Fish have extraor‐ dinarily sensitive and selective receptors that allow them to detect specific chemical signatures

Water participates in all life-essential molecular processes, from the most basic (e.g. Miller's experiment) to the most complex enzymatic processes, and it has allowed virtually all types of interatomic and molecular interactions that have resulted in the highly complex and diverse

The mineral world is massively involved in global geochemical processes, and in the compo‐ sition of solutes in the oceans, as salts and inorganic carbon sources. The cycling of elements is both tectonic and biogenic. Metals are essential catalysts of many organic reactions, and salts are actively involved in osmotic balance and membrane polarization. The availability of heteroatoms (P, S, halogens in particular) is (and has been originally) fundamental in the evolution of natural organic chemistry, because of their involvement in life-essential processes.

"Creative" chemistry is the work of living creatures, and Eubacteria and Archaea are probably the best organic chemists ever. Microbes are the drivers of global biogeochemical processes, and their genes have survived the great extinction events [28]. They have set the stage for other organisms to adapt and evolve. The capacity for pioneer organisms to modify their environ‐ ment for the benefit of other life forms is termed metabiosis, and it has been largely docu‐ mented in soil biology [29], but somewhat overlooked in aquatic ecosystems. They can occupy every niche of the biosphere, utilize different carbon and/or energy sources available in order

**4. Marine biodiversity and chemodiversity today**

to express itself ".

that influence their behavior.

chemical diversity observed in our oceans today.

**4.1. Minerals and geochemical cycles**

**4.2. Prokaryotes and prokaryote chemistry**

to generate ATP, the energy currency of all biochemical processes.

#### *Biodiversity explosions and mass extinctions (from [26])*

"Any lasting change in the biogeochemistry of any of the three components (atmosphere, seawater and land) will disrupt the interfacial equilibrium that supports the many thousands of life forms that interact constantly within an ecosystem. This has happened several times in the history of our planet since it became life-supporting. Changes in soil mineral strata indicate the occurrence of biodiversity-modifying events such as occupation by seas or the occurrence of an ice-age. Discrete organic layers may indicate the presence of a tropical rainforest or of a dry land savannah. Datable fossil evidence within these strata, together with paleontological reconstructions, point out the floristic and faunistic peculiarities of the times. Core drills in ice provide datable evidence of biogeoclimatic episodes within the last few millennia, while core drills in massive scleractinian corals give accurate calendar-like records of recurrent or of accidental climatic events affecting their biotope.

Speciation usually goes along with occupation of new territories and new habitats, the first colonizers having acquired the necessary adaptations to cope with evolving external demands – the Cambrian explosion (545 million years ago) being the most dramatic example of such adaptive diversification at all scales.

Along with this, evidences of accidental episodes of massive species extinctions are witnessed by the sudden "disappearance" of terrestrial and of marine life, that are attributable to tectonic, telluric or meteoritic impacts and to their profound and lasting climatologic and geochemical consequences. The most significant mass extinction is undoubtedly the Permian-Triassic Great Dying [23] where a 96% loss of all non-microbial marine life occurred within ten million years. The precise causes of mass extinction events may be in connection with continuous tectonic movements with their telluric and volcan‐ ic outbreaks and their climatic consequences, to collisions with meteoritic bodies, and to a lesser extent to the appearance of dominant predators, parasites or microbial diseases, or to combinations thereof. Common with many extinction events, however, is the massive release of greenhouse and of toxic gases (carbon dioxide, methane, hydrogen sulfide etc.). The water solubility of CO2 being nearly 30 times that of oxygen, water acidification occurs that impacts preferentially all calcifying organisms with low metabolic rates and weak respiratory systems: most coral genera died out during the Great Dying, along with calcareous sponges, calcifying algae, echinoderms, bryozoans etc. [24].

Interestingly, profound taxonomic changes in all major phyla seem to follow extinction events, resulting in a better adapted biodiversity. Nothing is known, however, on the consequence of such changes in microbial life or on the putative role microbial associates had on the *reinven‐ tion* (understand: adaptive evolution) of new species. As Falkowski [25] puts it, animals and plants are merely new incarnations of ancient metabolic processes, but the ultimate key to biodiversity may be held by bacteria ferrying the set of core genes that are necessary for life to express itself ".


**Author details**

Daniela Lazar1 \*, Sorina Taban2 , Marioara Cornianu2 , Alexandra Faur3 , Ioan Romosan4 and Adrian Goldis1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Gastroenterology, University of Medicine and Pharmacy "Victor Babeş" Timisoara, Romania

2 Department of Pathology, University of Medicine and Pharmacy "Victor Babeş" Timisoara, Romania

3 Department of Anatomy and Embryology, University of Medicine and Pharmacy "Victor Babeş" Timisoara, Romania

4 Department of Internal Medicine, University of Medicine and Pharmacy "Victor Babeş" Timişoara, Romania


**Inhibitory Effects of Roseoside and Icariside E4 Isolated from a Natural Product Mixture (No-ap) on the Expression of Angiotensin II Receptor 1 and Oxidative Stress in Angiotensin II-Stimulated H9C2 Cells**

**Eun Young Hong 1,†, Tae Yang Kim 2,†, Gwan Ui Hong 1 , Hanna Kang 2 , Jung-Yun Lee 3 , Jae Yeo Park 1 , Se-Chan Kim 4 , Young Ho Kim 5 , Myung-Hee Chung 6 , Young-In Kwon 2,\* and Jai Youl Ro 1,7,\***

Received: 21 December 2018; Accepted: 22 January 2019; Published: 23 January 2019

**Abstract:** Hypertension is a major risk factor for the development of cardiovascular diseases. This study aimed to elucidate whether the natural product mixture No-ap (NA) containing *Pine densiflora*, *Annona muricate*, and *Monordica charantia*, or its single components have inhibitory effects on hypertension-related molecules in Angiotensin II (Ang II)-stimulated H9C2 cells. Individual functional components were isolated and purified from NA using various columns and solvents, and then their structures were analyzed using ESI–MS, 1H-NMR, and 13H-NMR spectra. H9C2 cells were stimulated with 300 nM Ang II for 7 h. NA, telmisartan, ginsenoside, roseoside (Roseo), icariside E4 (IE4), or a combination of two components (Roseo and IE4) were administered to the cells 1 h before Ang II stimulation. The expression and activity of hypertension-related molecules or oxidative molecules were determined using RT-PCR, western blot, and ELISA. Ang II stimulation increased the expression of Ang II receptor 1 (AT1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), tumor growth factor-β (TGF-β) mRNA, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and the levels of hydrogen peroxide (H2O2) and superoxide anion (•O2 −) and reduced anti-oxidant enzyme activity. NA significantly improved the expression or activities of all hypertension-related molecules altered in Ang II-stimulated cells. Roseo or IE4 pretreatment either decreased or increased the expression or activities of all hypertension-related molecules similar to NA, but to a lesser extent. The pretreatment with a combination of Roseo and IE4 (1:1) either decreased or increased the expression of all hypertension-related molecules, compared to each single component, revealing a synergistic action of the two compounds. Thus, the combination of single components could exert promising anti-hypertensive effects similar to NA, which should be examined in future animal and clinical studies.

**Keywords:** cardiomyocytes (H9C2 cells); angiotensin II; hypertension; No-ap (natural product mixture); reactive oxygen species; roseoside; icariside E4

#### **1. Introduction**

Hypertension is a major risk factor for the development of cardiovascular diseases including coronary artery disease, stroke, heart failure, peripheral vascular disease [1]. Angiotensin II (Ang II) is a vasoactive peptide of the renin–angiotensin system (RAS). The cellular effects of Ang II are mediated by at least two receptors, Ang II receptor 1 (AT1) and Ang II receptor 2 (AT2). Ang II, through AT1 or AT2, plays a key role in blood pressure homeostasis [2]. Ang II binds AT1 to induce NADPH oxidase activation and leads to the production of reactive oxygen species (ROS) [3–5]. Thus, the increasing of ROS is involved in cardiovascular diseases-related changes such as hypertrophy, fibrosis, tissue inflammation, or vascular remodeling in the heart [4–6].

Inhibition of the RAS through AT receptor blockers (ARBs) can prevent cardiovascular disease-related events [7]. ARBs (for example, losartan and telmisartan), which are a new class of approved anti-hypertensive agents, prevent the hypertensive effects of Ang II by the selective blockade of AT1 [8,9]. However, ARBs produce undesirable side effects such as headache, fatigue, and dizziness [7]. Thus, we have much interest in the search for natural products with anti-hypertensive effects and reduced side effects.

Our company (Hyunsung Vital Co. Ltd., Seoul, Korea) has manufactured a natural product complex (No-ap, NA) expected to downregulate blood pressure. NA contains three natural materials, i.e., *Pinus densiflora*, *Annona muricata* L., and *Monordica charantia*.

*P. densiflora* is widely distributed around the world, particularly in Korea and Japan [10]. Pine bark or needle have been reported to be effective scavengers of ROS [11,12] and to have a suppressive effect on the expression of pro-inflammatory mediators [13]. Pine needles have anti-hypertensive effects [14]. *A.a muricata* L., which is known as graviola or guanabana, is widely found in India, South and Central America, tropical West Africa, and Asia [15]. It has been reported that a decoction made from *A. muricata* can be used for hypertension therapy and that the plant extract has anti-oxidative and anti-hypertensive properties [16]. *M. charantia* L. is a common vegetable in Okinawa, where it has been recently used in the therapy of hypertension, diabetes, and dyslipidemia [17], and its phenolic extract has inhibitory properties against angiotensin-1-converting enzyme, hypertension, and oxidative stress [18]. Thus, these findings imply that NA may have anti-hypertensive effects, as it is also supported by its wide use as a folk remedy and by laboratory experiments [14–16].

Although there are reports that the different constituents of NA, i.e., *P. densiflora* needle, *A. muricata*, and *M. charantia* may have anti-hypertensive properties, the single components of this mixture have not been isolated and examined [14–16]. This study investigated for the first time whether NA has inhibitory effects on the hypertension-related molecules in Ang II-stimulated H9C2 cells. After confirmation of the anti-hypertensive effects, this study aimed to identify the single functional components of NA and to investigate whether they have anti-hypertensive properties individually. We observed that the pretreatment with a combination of roseoside and icariside E4, which showed strong activity among the five single components identified in NA, had anti-hypertensive effects by downregulating ROS generated via the expression of AT1 and the activity of NADPH oxidase.

#### **2. Results**

#### *2.1. Effects of NA on the Expression of Hypertension-Related Molecules in Ang II-Stimulated H9C2 Cells*

AT1 is an important effector controlling blood pressure (BP) and blood volume in the cardiovascular system [3]. We first examined the effects of NA on AT1 expression in Ang II-stimulated H9C2 cells. AT1 expression was increased in the Ang II-stimulated H9C2 cells, compared with negative control

(NC, treated with phosphate-buffered saline) cells. NA (60, 100, 200 µg/mL) reduced AT1 expression in a dose-dependent manner (Figure 1A). A high dose of NA reduced AT1 expression similar to telmisartan (Telmis), which is known as an AT1 blocker preventing Ang II-induced oxidative stress and vascular remodeling in hypertension [9]. Pretreatment with 200 µg/mL (corresponding to the high dose of NA) of ginsenoside (Gin), which was used as one of the natural positive controls for the natural product mixture (NA), had no effect on AT1 expression in Ang II-stimulated H9C2 cells.

**Figure 1.** Effects of the natural product mixture (No-ap, NA) on the expression of hypertension-related molecules or oxidative stress in the Ang II-stimulated H9C2 cells. H9C2 cells (1 × 106 cells) were stimulated with 300 nM Ang II for 7 h. No-ap (NA), telmisartan (Telmis), or ginsenoside (Gin) were administered 1 h before Ang II stimulation. The expression of AT1, TNF-α, MCP-1, TGF-β was determined in mRNA extracts isolated from H9C2 cells using RT-PCR. The activity of NADPH oxidase, catalase, and SOD, and the amounts of H2O2 and •O2 − were determined in cell lysates isolated from H9C2 cells using an ELISA kit. The reactions were analyzed using an ELISA plate reader at 450 nm for the activities of NADPH oxidase and SOD and •O2 − amounts, and at 590 nm for H2O2 amounts and catalase activity. (**A**) Expression of AT1 and cytokines. (**B**) Activity of NADPH oxidase. (**C**) Amounts of H2O2 . (**D**) Amounts (fold change %) of •O2 −. (**E**) Activities of catalase and SOD. #, Numbers below the band images, indicating the mean values (*n* = 4 independent experiments) obtained from the ratio of the band density of each group to those of the corresponding controls and loading control GAPDH. The results represent the mean ± SEM (*n* = 4) obtained from four independent experiments performed in triplicates. NC, negative control; Ang II, angiotensin II stimulation; AT1, angiotensin II receptor 1; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein-1; TGF-β, tumor growth factor-β; NADPH, nicotinamide adenine dinucleotide phosphate; H2O2 , hydrogen peroxide; •O2 −, superoxide anion; SOD, superoxide dismutase. \*\*\*, *p* < 0.001 versus the NC. + , *p* < 0.05; ++ , *p* < 0.01; +++ , *p* < 0.001 versus the Ang II stimulation.

It has been reported that inflammation has a crucial role in the pathogenesis of hypertension [19–21]. The inflammatory process, with ROS generation and increase in cytokines' releases, is a hallmark of hypertension [20,21]. Thus, in order to investigate whether NA prevents inflammation in Ang II-stimulated H9C2 cells, the expression of inflammatory cytokines was examined. The expression levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and tumor

growth factor-β (TGF-β) were increased in Ang II-stimulated H9C2 cells (Figure 1A). NA pretreatment significantly suppressed the expression of these inflammatory cytokines caused by Ang II in a dose-dependent manner. A high dose of NA showed stronger inhibitory responses than Telmis. Gin did not show any effects in all cases. Therefore, hereafter, we will not consider Gin effects.

#### *2.2. Effects of NA on Oxidative Stress in Ang II-Stimulated H9C2 Cells*

It has been reported that Ang II-induced hypertension by ROS generated via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [22,23]. Thus, we examined the effects of NA on NADPH oxidase activity in Ang II-stimulated H9C2 cells. NA pretreatment reduced NADPH oxidase activity in Ang II-stimulated H9C2 cells (Figure 1B).

Next, we examined the effects of NA on the generation of ROS or on anti-oxidant enzyme activity in Ang II-stimulated H9C2 cells. The production of hydrogen peroxide (H2O2) or superoxide anion (•O2 −) increased by Ang II stimulation was diminished in NA-pretreated cells in a dose-dependent manner (Figure 1C,D). The activities of catalase or superoxide dismutase (SOD) were decreased in Ang II-stimulated H9C2 cells. NA pretreatment increased anti-oxidant enzyme activity (catalase and SOD) in Ang II-stimulated H9C2 cells in a dose-dependent manner, similar to Telmis (Figure 1E).

#### *2.3. Purification and Identification of Bioactive Ingredients in NA*

As a natural product mixture, NA contains *P. densiflora* (75.0%), *A. muricata* (12.5%), *M. charantia* (12.5%). NA was extracted with methanol and successively partitioned with ethyl acetate and n-BuOH to afford ethyl acetate, n-BuOH, and water fractions. The n-BuOH fraction was subjected to silica gel and YMC RP-18 silica gel column chromatography, and five compounds (**1**–**5**) were identified. Their spectroscopic data and comparisons with previous data confirmed that these compounds were roseoside (**1**) [24], isolariciresinol 9-*O*-β-D-xyloside (**2**) [25], massonianoside B (**3**) [26], icariside E4 (**4**) [27], and nicotiflorin (**5**) [28] (Figure 2, Supplementary Materials Figures S1–S15).

**Figure 2.** Chemical structures of the compounds isolated from NA. NA (1.9 kg) was fractioned using various organic chemicals and columns as described in "Materials and Methods". Finally, five single components were identified from NA, and then the structure of each component was analyzed using ESI–MS, 1H-NMR, and 13C-NMR spectra. The inhibitory effects on the expression of AT1 of each fraction separated with organic chemicals were determined (data not shown). Structures of (**1**), (**2**), (**3**), (**4**), and (**5**) indicate roseoside, isolariciresinol 9-*O*-β-D-xyloside, massonianoside B, icariside E4, and nicotiflorin, respectively. The molecular formulas of the components roseoside and icariside E4, which demonstrated biological activity, re C19H30O8 (MW, 386.1941) and C26H34O10 (MW, 506.5480), respectively.

#### *2.4. Effects of the Isolated Components Roseoside and Icariside E4 on the Expression of Hypertension-Related Molecules in Ang II-Stimulated H9C2 Cells*

We first confirmed that two single components [roseoside (Roseo) and icariside E4 (IE4)], among the five isolated components, had biological activity. Three components (isolariciresinol 9-*O*-β-D-xyloside, massonianoside B, and nicotiflorin) did not have any effects on the expression of AT1 (data not shown). Roseo or IE4 (20, 30, 50 µg/mL) pretreatment significantly reduced the expression of all hypertension-related molecules (mRNA and protein) in Ang II-stimulated H9C2 cells in a dose-dependent manner, compared to Ang II stimulation alone (Figure 3). Treatment with 70 µg/mL of Roseo or 100 µg/mL of IE4 reduced the expression of all hypertension-related molecules by approximately 48% and 50%, respectively, compared to Ang II stimulation. However, only Roseo showed downregulating activity at a dose above 70 µg/mL (data not shown). Both components showed inhibitory effects on the expression of all hypertension-related molecules, although neither one exhibited inhibitory effects similar to those of Telmis or NA. Thus, we tried a combination pretreatment. A ratio of 1:1 for the combination pretreatment of Roseo and IE4 was used in preliminary experiments. The combination of Roseo and IE4 in a 1:1 ratio (each used in a dose of 25 µg/mL; total dose, 50 µg/mL) reduced the expression of all hypertension-related molecules by approximately 50–72% in Ang II-stimulated H9C2 cells. Thus, the combination pretreatment of Roseo and IE4 showed a strong synergistic inhibitory effect on the expression of all hypertension-related molecules in Ang II-stimulated H9C2 cells.

**Figure 3.** Effects of roseoside and icariside E4, alone or in combination, on the expression of hypertension-related molecules in the Ang II-stimulated H9C2 cells. The experimental details of the stimulation of H9C2 cells and their treatment with different compounds were described in Figure 1 legend. Telmis (10 µM), roseoside (Roseo; 20, 30, or 50 µg/mL), icariside E4 (IE4; 20, 30, or 50 µg/mL), and Roseo/E4 combinations (20, 30, or 50 µg/mL, 1:1 ratio) were administered 1 h before Ang II stimulation. The expression of all hypertension-related molecules was determined in mRNA or protein extracts isolated from H9C2 cells using RT-PCR and Western blot, respectively. (**A**) Expression of all hypertension-related molecules' mRNA. (**B**) Protein expression of all hypertension-related molecules. Both, combination of Roseo and IE4 (1:1 ratio; each component used at a dose of 10, 15, 25 µg/mL; total doses for the different mixtures at a 1:1 ratio were 20, 30, 50 µg/mL). Ang II, angiotensin II stimulation. #, Numbers below the band images indicate the mean values (*n* = 4) obtained as described in Figure 1 legend.

#### *2.5. Respective Effects of Roseoside and Icariside E4 on Oxidative stress in Ang II-Stimulated H9C2 Cells*

We examined the effects of Roseo or IE4 on NADPH oxidase activity in Ang II-stimulated H9C2 cells. Roseo or IE4 pretreatment reduced NADPH oxidase activity in Ang II-stimulated H9C2 cells (Figure 4A).

**Figure 4.** Effects of roseoside, icariside E4, and their combination on oxidative stress in Ang II-stimulated H9C2 cells. The experimental details of the stimulation of H9C2 cells and their treatment with different compounds were described in Figure 1 legend. Telmis (10 µM), Roseo (20, 30, or 50 µg/mL), IE4 (20, 30, or 50 µg/mL), and combinations of Roseo and IE4 (20, 30, or 50 µg/mL, 1:1 ratio) were administered to the cells 1 h before Ang II stimulation. The activities of NADPH oxidase, catalase, and SOD, and the amounts of H2O2 and •O2 − were determined in cell lysates isolated from H9C2 cells using an ELISA kit. The reactions were analyzed using an ELISA plate reader as described in Figure 1 legend. (**A**) Activity of NADPH oxidase. (**B**) Amounts of H2O2 . (**C**) Amounts (fold change %) of •O2 −. (**D**) Activities of catalase and SOD. The results represent the mean ± SEM (*n* = 4) obtained from four independent experiments performed in triplicates. \*\*\*, *p* < 0.001 versus NC. + , *p* < 0.05; ++ , *p* < 0.01; +++ , *p* < 0.001 versus Ang II stimulation.

Next, we examined the effects of Roseo and IE4 on the generation of ROS and on anti-oxidant enzyme activity in Ang II-stimulated H9C2 cells. The generation of H2O2 or •O2 − increased by Ang II stimulation was diminished after Roseo or IE4 pretreatment in a dose-dependent manner (Figure 4B,C). Both Roseo and IE4 pretreatments increased the anti-oxidant activity (catalase and SOD), which was decreased by Ang II-stimulated H9C2 cells (Figure 4D). Roseo pretreatment showed reduction of ROS and increase of anti-oxidant enzyme activity similar to Telmis, whereas IE4 pretreatment showed smaller effects than Roseo or Telmis.

The combination pretreatment of Roseo and IE4 at a ratio of 1:1 (each used at the dose of 25 µg/mL; total dose, 50 µg/mL) reduced the activity of NADPH oxidase and the generation of H2O2 and •O2 − in a dose-dependent manner in Ang II-stimulated H9C2 cells. The combination pretreatment increased the activity of catalase or SOD in a dose-dependent manner. Thus, the combination pretreatment

of Roseo and IE4 at a 1:1 ratio showed stronger synergistic inhibitory effects on the suppression of oxidative stress and on the increase of anti-oxidant enzyme activities in Ang II-stimulated H9C2 cells, compared to the single components (Figure 4).

#### **3. Discussion**

We demonstrated that the natural product complex NA suppresses the expression of AT1, the generation of ROS (H2O2 and •O2 −), and the expression of inflammatory cytokines (TNF-α, MCP-1, and TGF-β) produced via NADPH oxidase, which are related to hypertension, and that it increases the expression of anti-oxidant enzymes (catalase and SOD) in Ang II-stimulated H9C2 cells. Our data also demonstrate that two components, Roseo and IE4, among five identified components isolated and purified from NA, whose structures were also analyzed in this study, have functional activities in hypertension.

The component Roseo isolated from various plants, including *A. muricata* contained in NA, has a variety of functional activities. It relaxes precontracted aortic rings in an endothelium-dependent manner [29], increases insulin secretion [30], inhibits rat liver microsomal glucose-β-phosphate [31], potentiates the inhibitory activity against angiotensin-converting enzyme, although itself shows no activity against an its enzyme [32], shows inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells [33], prevents oxidative stress [34], and has a depigmentation effect in melanocytes by inhibiting melanin synthesis [35]. However, it has not been reported yet that Roseo directly suppresses hypertensive effects caused by the upregulation of ROS.

The other component, IE4, isolated and purified from plants, such as *Ulmus pumila* L. and *Tabebuia roseo-alba*, has various functional activities. It inhibits NO production [36], shows anti-oxidant activity [37], demonstrates anti-nociceptive activity in a chemical pain-induced model [38], and may be beneficial in the traditional treatment of Alzheimer's disease by preventing blood–brain barrier damage and inflammatory cell infiltration into the brain [39]. IE4, purified from pine trees, such as *P. densiflora*, *Pinus thunbergii*, and *Pinus morrisonicola* Hayata, also delays the coagulation time by inhibiting thrombin activity [40] and inhibits the release of β-hexosaminidase in RBL-2H3 cells [41]. However, it has not been reported yet that IE4 purified from *P. densiflora*, which is the most abundant component of NA, directly suppresses the hypertensive effects caused by the upregulation of ROS. There is only a report indicating that IE4 isolated from *P. morrisonicola* H. could be a promising anti-hypertensive candidate by blocking voltage-operating Ca2+ channels [42].

Hypertension is the most common cardiovascular risk factor. Ang II, which is known as one of many factors causing cardiovascular injury in hypertension, elicits many pathophysiological actions by inducing ROS generation via the activation of vascular NADPH oxidase [20,22,23,43]. Ang II stimulation can increase blood pressure in association with immune response activation and inflammation [21]. Infiltration of inflammatory cells into areas around blood vessels that occurs simultaneously with other events of the inflammatory process, such as the increase of ROS generation and of the levels of cytokines and chemokines, is a hallmark of hypertension [9,21,22]. Thus, our data suggest that NA may suppress blood pressure and vascular remodeling in hypertension mainly caused by RAS through downregulating of ROS produced via AT1 expression and NADPH oxidase activity, as demonstrated by the data showing that NA reduced hypertensive responses through inhibiting downstream pathways via AT1 expression and NADPH oxidase (which generates ROS), and then the generated ROS increased the release of the cytokine TNF-α and the chemokines MCP-1, which is known to induce inflammation and cell infiltration [44], respectively, and increased TGF-β, which is known to induce vascular remodeling in hypertension [9,22]. In addition, our data indicate that NA increased endogenous anti-oxidant enzymes (catalase and SOD) in Ang II-stimulated H9C2 cells. The endogenous anti-oxidant glutathione peroxidase (GPx), which scavenges H2O2, is not affected by Ang II stimulation in cardiac fibroblasts [45] and vascular adventitial fibroblasts [46]. Thus, we did not check GPx activity because the catalase also scavenges H2O2. However, it is necessary to investigate the differences between cell types.

Our observations also suggest that the components Roseo and IE4, isolated and purified from NA, may independently suppress hypertension-related molecules such as ROS, cytokines and chemokines, and TGF-β, which are typically associated with oxidative stress, inflammation, and vascular remodeling in hypertension, by increasing endogenous anti-oxidant enzymes in Ang II-stimulated H9C2 cells. In addition, we also show that combinations of Roseo and IE4 (1:1 ratio) may have strong synergistic or additive effects.

In conclusion, these in vitro results support the conclusion that the functional compounds Roseo and IE4 have significant anti-hypertensive effects on Ang II-stimulated H9C2 cells. Our data suggest that Roseo and IE4 from NA, which have anti-oxidant, anti-inflammatory, and anti-vascular remodeling properties in hypertension and less side effects than the whole mixure NA, have a potential as health functional food supplements for hypertension and should be further evaluated in animal and clinical models.

#### **4. Materials and Methods**

#### *4.1. Materials*

NA, a natural product mixture, was donated by Hyunsung Vital Co. Ltd. (Seoul, Korea) which makes a variety of healthy functional foods. NA contains *P. densiflora* (75.0%), *A. muricata* (12.5%), and *M. charantia* (12.5%). These plants were extracted using hot water (90 ◦C) for 24 h, and the extract was evaporated by a Liquefied extractor (Hyunsung Vital Co. Ltd., Seoul, Korea) to yield a powder of NA. This powder was named NA (No-ap), natural product mixture.

#### *4.2. Purification and Identification of Bioactive Ingredients*

NA (1.9 kg) was extracted using methanol (4 L, 95%) at room temperature for 2 days. The methanol extract (556.4 g) was concentrated under pressure, dissolved in distilled water (2 L), and successively partitioned with ethyl acetate and n-BuOH to afford ethyl acetate (23.0 g, A), n-BuOH (122.1 g, B), and water fractions.

The n-BuOH extract (120 g) was separated by vacuum liquid chromatography using a silica gel column with a gradient solvent mixture of CHCl3–MeOH (30:1, 25:1, 20:1, 15:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 100% MeOH) to afford 11 subfractions (B-1 to B-11). Next, subfraction B-6 (2.1 g) was subjected to YMC RP-C18 silica gel column and was eluted with a solvent mixture of MeOH–H2O (1:2), yielding compound 1 (20.0 mg), compound 2 (10.0 mg), compound 3 (30.0 mg), and compound 4 (10.0 mg). Further purification of subfraction B-9 via YMC RP-C18 silica gel column, using mixtures of MeOH−H2O (1:1.5), and preparative HPLC yielded compound 5 (10.0 mg).

#### *4.3. Cardiomyocytic H9C2 Cell Line Culture*

The rat cardiomyocytic H9C2 cell line (H9C2 cells) was obtained from the Korean Cell line Bank (KCLB, Seoul, Korea). H9C2 cells were grown in Dulbecco's modified eagle medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 1% L-glutamine, 1% antibiotic penicillin/streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (HyClone, Logan, UT, USA). H9C2 cells were maintained at 37 ◦C in a humidified atmosphere with CO2, and the media were replaced every 3 days [47].

#### *4.4. Cell line Stimulation and Treatment*

H9C2 cells (1 × 106 cells) were stimulated with 300 nM angiotensin II (Ang II; Sigma-Aldrich, St. Louis, USA) and then incubated for 7 h [47]. The cells were centrifuged (470× *g*, 3 min) to separate the supernatants and pellet the cells. The cells were used to determine the expression of all molecules related to hypertension. NA (60, 100 or 200 µg/mL), telmisartan (Telmis; 10 µM), ginsenoside (Gin; 200 µg/mL), roseoside (Roseo; 20, 30 or 50 µg/mL), icariside E4 (IE4; 20, 30 or 50 µg/mL), the last two as purified components isolated from NA, and combinations of Roseo and IE4 (1:1 ratio,

20, 30, or 50 µg/mL) were administered to the cells 1 h before Ang II stimulation. As a negative control (NC), phosphate-buffered saline (PBS) was used, which was also Ang II solvent. Telmis, which is a drug used in the clinic, was used as a positive control. The optimal concentrations of Ang II stimulation, NA, Gin, Roseo, and IE4 were determined in preliminary experiments (data not shown). Gin was used as a natural positive control for the natural product mixture (NA).

#### *4.5. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)*

Total mRNA was isolated from H9C2 cells (1 × 106 cells) using TRIzol reagent (Invitrogen, Life Technologies Ltd., Waltham, CA, USA). RT-PCR was performed in a final volume of 20 µL, using a high-capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) and a G-taq kit (Cosmogenetech, Seoul, Korea) in an automated thermal cycler (Bio-Rad, Laboratories, Hercules, CA, USA). The PCR assays were performed for 35 cycles. Each cycle consisted of the following steps: denaturation at 94 ◦C for 30 s, annealing at 51 ◦C for 45 s, and extension at 72 ◦C for 1 min. The results were expressed as a ratio to GAPDH mRNA. The PCR products were analyzed using 1% agarose gel and visualized under UV light after staining with StaySafe nucleic acid gel stain (Real Biotech Corporation, Banqiao, Taiwan) [48].

The primer sequences used were as follows: AT1 sense, 50 -CAT AGG ACT GGG CCT AAC CA-30 ; AT1 anti-sense, 50 -GCC GTA AAA CAG AGG GTT CA-30 ; TNF-α sense, 50 -TTC TGT CCC TTT CAC TCA CTG G-30 ; TNF-α anti-sense, 50 -TTG GTG GTT TGC TAC GAC GTG G-30 ; MCP-1 sense, 50 -GAA GGA ATG GGT CCA GAC AT-30 ; MCP-1 anti-sense, 50 -ACG GGT CAA CTT CAC ATT CA-30 ; TGF-β sense, 50 -CTC TCC ACC TGC AAG ACC AT-30 ; TFG-β anti-sense, 50 -CTG CCG TAC AAT TCC AGT GA-30 ; GAPDH sense, 50 -AAC TTT GGC ATT GTG GAA GG-30 ; GAPDH anti-sense, 50 -ACA CAT TGG GGG TAG GAA CA-30 .

## *4.6. Determination of the Activity of NADPH Oxidase, Superoxide Dismutase, and Catalase and the Levels of Hydrogen Peroxide and Superoxide Anion*

The activities of NADPH oxidase, catalase, and SOD, and the amounts of H2O2 and •O2 − were measured in the lysates of H9C2 cells stimulated with Ang II using a NADPH oxidase assay kit, a H2O2 assay kit (Abcam, Cambridge, UK), a •O2 − assay kit, a SOD assay kit (Cell Biolabs. Inc., San Diego, CA, USA), and a catalase activity kit (Biovision, Milpitas, CA, USA), respectively. Briefly, H9C2 cells (1 × 106 cells) were washed three times with PBS and placed in lysis buffer (PBS containing 1% Triton X-100). Standard diluents (100 µL), lysates obtained from each sample (100 µL), and 100 µL of reaction mixture (50 µL enzyme working solution and 50 µL probe) were added in 96-well plates; the plates were then incubated on a plate shaker at room temperature for the time periods indicated (30 min for NADPH oxidase, SOD, and catalase; 1 h for H2O2 and •O2 −). The optical density was read at 450 nm for NADPH oxidase, •O2 −, and SOD, and at 590 nm for H2O2 and catalase. Standard curves were made using serial dilutions of a standard sample, and then the activity was calculated according to the manufacturer's instructions. The lowest detection limit for NADPH oxidase was below 20 pg/mL, for H2O2 it was 0.04 pmol/µL, for •O2 − and SOD it was 1.2 mU/µL, and for catalase it was 0.078 pg/mL.

#### *4.7. Western Blot Analysis*

H9C2 cells (1 × 106 cells) harvested from Ang II-stimulated cells were suspended in a low-salt lysis buffer [50 mM Tri-HCl (pH 7.9), 1.0 mM EDTA, 150 mM NaCl, 1.0% NP40, 5 mM NaF, 0.25% Na deoxycholate, 2 mM NaVO3, protease inhibitors cocktail] and allowed to swell on ice for 30 min. The cells were then homogenized using a micropipette. After centrifugation, the supernatants obtained from the cell extracts were analyzed by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were washed with PBS containing 0.1% Tween 20 (PBST) and then blocked for 1 h in PBST containing 5% skim milk. After washing the membranes with PBST, they were treated with primary antibodies against actin, AT1, TNF-α (Cell Signaling

Technology, Beverly, MA, USA), MCP-1, or TGF-β (Abcam, Cambridge, UK) diluted with PBST (1:1000). The membranes were washed with PBST and treated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit IgG (diluted to 1:5000) (Bethyl Laboratories, Montgomery, TX, USA) in PBST for 1 h. After washing, the protein bands were visualized by Enhanced Chemi-Luminescence (ECL; Amersham Biosciences, Piscataway, NJ, USA) using a chemiluminometer (Bio-Rad, Laboratories, CA, USA) [48].

#### *4.8. Statistical Analysis*

The experimental data are shown as means ± SEM (*n* = 4). The unpaired Student's *t*-test was used to compare two groups. Multiple-group comparisons were performed using two-way ANOVA followed by Scheffe's post-hoc test, using the SPSS software (SPSS Inc., Chicago, IL, USA). Values of *p* < 0.05 were considered to indicate statistical significance. Densitometry analyses of Western blots and RT-PCR were performed with Quantity One (version 4.6.3; Bio-Rad, Hercules, CA, USA) and the results are indicated as means ± SEM (*n* = 4), obtained from the ratio of each band density to those of the control and the loading control of four independent experiments.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/3/414/s1, Figure S1: 1H-NMR spectra of roseoside (**1**), Figure S2: 13C-NMR spectra of roseoside (**1**), Figure S3: ESI–MS spectra of roseoside (**1**), Figure S4: 1H-NMR spectra of (−)-marmesinin (**2**). Figure S5: 13C-NMR spectra of isolariciresinol 9-*O*-β-D-xyloside (**2**), Figure S6: ESI-MS spectra of isolariciresinol 9-*O*-β-D-xyloside (**2**), Figure S7: 1H-NMR spectra of massonianoside B (**3**), Figure S8: 13C-NMR spectra of massonianoside B (**3**), Figure S9: ESI-MS spectra of massonianoside B (**3**), Figure S10: 1H-NMR spectra of icariside E4 (**4**), Figure S11: 13C-NMR spectra of icariside E4 (**4**), Figure S12: ESI-MS spectra of icariside E4 (**4**), Figure S13: 1H-NMR spectra of nicotiflorin (**5**), Figure S14: 13C-NMR spectra of nicotiflorin (**5**), Figure S15: ESI-MS spectra of nicotiflorin (**5**).

**Author Contributions:** J.Y.R. designed the study and directed its implementation, including the study's analytic strategy, and prepared the Discussion section of the text. Y.-I.K. helped conduct the literature review and prepare the Methods and the Discussion sections of the text. Y.H.K., S.-C.K., J.-Y.L., H.K., and T.Y.K. prepared the products to be tested and participated in the preparation of the manuscript. M.-H.C. helped conduct the literature review and designed the study. E.Y.H., G.U.H., and J.Y.P. conducted the cell experiments and analyzed the data. All the authors read and approved the final manuscript.

**Funding:** This study was supported by a research grant from Life & Science Research Center, Hyunsung Vital Co. Ltd., Seoul, Korea 07255, and from Hannam University in 2018, Daejeon 34054, Korea.

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

#### **References**

48. Hong, G.U.; Kim, N.G.; Kim, T.J.; Ro, J.Y. CD1d expressed in mast cell surface enhances IgE production in B cells by up-regulating CD40L expression and mediator release in allergic asthma in mice. *Cell. Signal.* **2014**, *26*, 1105–1117. [CrossRef]

**Sample Availability:** Samples of the compounds are available from the corresponding authors (Y.-I.K.; J.Y.R.).

© 2019 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/).


**2. Acute pancreatitis**

AP is an acute inflammatory condition of the pancreas with histological acinar cells destruction. It has a wide spectrum of morphological and clinical manifestations and can result in local injury, systemic inflammatory response syndrome (SIRS) and organ failure [1, 2].

### **2.1 Epidemiology**

AP is one of the most common gastrointestinal diseases requiring acute hospitalization [3]. Its incidence is rising worldwide and ranges from 5 to 30 cases per 100,000 [1] and despite improvements in the diagnosis, management and treatment, the overall mortality rate of AP remains around 2–5% [4, 5]. The average length of hospital stay for AP is 8 days, with economic burden to patients and the health care system all around the world [6].

### **2.2 Etiology**

The most common causes of AP are gallstones (up to 40–70% of cases) and alcohol abuse (25–35%).

Migrating gallstones cause transient obstruction of the pancreatic duct leading to the blockage of pancreatic secretion and lysosomal dysfunction generating injury and inflammatory response. Alcohol abuse exerts its effects in a complex way that include direct toxicity and immunologic mechanisms: prolonged alcohol use (four to five drinks in a day over a period of more than 5 years) is required and the type of alcohol ingested does not affect the overall lifetime risk of alcohol-associated pancreatitis, that range from 2% to 5% in heavy drinkers ("Heavy" alcohol consumption is generally considered to be >50 g in a day).

In absence of gallstones or alcohol, other etiologies of AP (**Table 1**) must be ruled out.

The agent or condition causing AP is not always clear and sometimes there is only the evidence of factors known to be potential contributors of unexplained pancreatitis, such as smoke, obesity and diabetes. Accordingly, idiopathic AP has been defined as a condition in which the etiological cause is not detectable after an accurate anamnesis excluding any substance abuse, infections, metabolic disorders, genetic mutations and at least two second-level imaging techniques [endoscopic ultrasound and

*Management of Acute and Chronic Pancreatitis DOI: http://dx.doi.org/10.5772/intechopen.109115*

*\*Among patients undergoing ERCP.\*\*5–14% of patients with benign or malignant pancreatobiliary tumors present with AP.*

### **Table 1.**

*Causes of acute pancreatitis.*

magnetic resonance imaging (MRI)] to exclude abnormality of pancreatic gland, pancreatic or biliary and gallbladder lithiasis.

Any mass that obstructs the main pancreatic duct can cause AP: 5–14% of patients with benign or malignant pancreatobiliary tumors present with this scenario and pancreatic tumor should be suspected in any patient older than 40 years with idiopathic pancreatitis, especially those with prolonged or recurrent course [4–6].

### **2.3 Clinical signs and symptoms**

Patients with AP usually present with epigastric or left upper quadrant pain, usually described as persistent, severe and often radiating to the back, chest or flanks. The intensity of pain is not correlated to the severity of the disease. Patients experience pain relief when sitting forward or worsening when lying flat. Nausea and vomiting are also common, and sequestered fluid in the small bowel may lead to rapid and severe dehydration. Diaphragmatic irritation may cause hiccoughs. The presentation can also be dominated by shock with tachycardia, tachypnea, hypotension, anuria and mental status alteration. On the other hand, patients may be paucisymptomatic,

with few physical signs. Abdominal examination reveals epigastric tenderness and guarding; abdominal distension with paralytic ileus. Later signs may include mottled skin or livedo reticularis and lace-like purplish discoloration of the skin. Abdominal periumbilical ecchymosis (Cullen's sign) and ecchymosis of the flank (Grey Turner's sign) result from the diffusion of fat necrosis and inflammation associated with retroperitoneal or intra-abdominal bleeding [5].

### **2.4 Diagnosis**

The diagnosis of AP is made following the Revised Atlanta Criteria, a global consensus classification (generated in 1992 and revised in 2012) designed to standardize diagnosis, clinical assessment, evaluation, severity and complications of AP and to help the communication between clinicians.

Diagnosis of AP requires two of the following three features:

According to these criteria, it is important to underline that when the diagnosis of AP is established by abdominal pain and by increased serum pancreatic enzyme activities (clinical and laboratory criteria), the radiological findings (imaging criteria) are not required for making the diagnosis [3, 7].

In the majority of patients, routine use of CT or MRI is unwarranted as the diagnosis of AP is apparent and most have a mild, uncomplicated course. CT or MRI imaging should be reserved for patients in whom the diagnosis is unclear or who fail to improve clinically within the first 48–72 hours after hospital admission [6].

Contrarily, transabdominal ultrasound should be performed on admission in all patients with AP, to define the underlying etiology and to identify the presence of gallstones that are the most common cause of AP [3, 6].

Moreover, it is important to record the time interval between onset, first observation and hospital admission. In fact, the onset of AP is defined as the time of beginning of abdominal pain and not the time of admission to the hospital [7].

In an episode of AP, the enzyme secreted by the pancreas (amylase, lipase, elastase and trypsin) are released from acinar cells of the pancreas into the bloodstream at the same time, due to increased permeability following inflammation.

Amylase is an enzyme synthesized mostly by pancreatic acinar cells and salivary glands and in negligible levels by adipose tissue, gonads, fallopian tubes, intestinal tract and skeletal muscle. Humans product one specific isoenzyme, α-amylase, with two major isoforms specific to pancreas and to salivary glands that help to identify different cases of hyperamylasemia. In case of AP, serum amylase rises rapidly within a few hours after the onset, with peaks at 3–6 hours, half-life of 10–12 hours, persistent elevation for 3–5 days and decrease to normal levels over the next three to 7 days.

Lipase is an enzyme that has a higher specificity because is mainly produced by acinar cells of the pancreas; nevertheless, high serum level can be determined also in patient with renal insufficiency, appendicitis, diabetic ketoacidosis, inflammatory

bowel disease and intestinal obstruction. In AP, elevation of serum lipase arises within three to 6 hours with peaks at 24 hours following the onset of symptoms and persistent elevation up to 2 weeks, giving a larger diagnostic window in comparison to amylase.

Therefore, serum lipase appears to be more specific and remains elevated for a longer period than serum amylase after disease presentation. Moreover, lipase has a better degree of sensitivity and specificity in diagnosing AP, during both early and late phases of the disease (sensitivity of lipase and amylase tests ranges between 64–100% and 45–87%, respectively).

According to these evidences, current guidelines recommend the preference use of serum lipase for diagnosis of AP [2, 4, 6].

### **2.5 Classification**

The most commonly used classification system for AP is the "2012 revision of the Atlanta Classification and definitions" based on international consensus [8].

This classification identifies two types (Interstitial edematous pancreatitis and necrotizing pancreatitis), three grades of severity (mild, moderately severe or severe) and two phases (early and late) of AP.

### *2.5.1 Types of acute pancreatitis*

Two different types of AP have been characterized: Interstitial edematous pancreatitis and necrotizing pancreatitis.

**Interstitial edematous pancreatitis** is an acute inflammation of pancreatic parenchyma and peri-pancreatic tissues, but without recognizable tissue necrosis. Developed by the majority of patients (80–85%), it is characterized by diffuse (or occasionally localized) enlargement of the pancreas, due to inflammatory edema; the clinical symptoms usually resolve within the first week.

**Necrotizing pancreatitis** is, instead, the presence of inflammation associated with pancreatic parenchymal necrosis and/or peri-pancreatic necrosis. The natural history of necrotizing pancreatitis is variable and this scenario evolves over several days because necrosis can remain solid or liquefy, can remain sterile or become infected, persist or disappear over time. This explains why an early CT made for assessment of AP may underestimate the eventual extent of pancreatic and peri-pancreatic necrosis. Moreover, most evidence suggest no correlation between the extent of necrosis and the risk of infection and duration of symptoms and usually infected necrosis is rare during the first week. Developed by 15–20% of patients with AP, this type of evolution of AP has increased morbidity and mortality compared to patients with interstitial edematous pancreatitis [5, 7].

### *2.5.2 Severity of acute pancreatitis*

A preliminary overview of complications of AP is mandatory, because the comprehension of these terminologies is central to definition and stratification of severity.

• **Local complications**: acute peri-pancreatic fluid collection, pancreatic pseudocyst, acute necrotic collection (sterile or infected), walled of necrosis (sterile or infected), gastric outlet dysfunction, splenic and portal vein thrombosis, ischemic colitis, colonic necrosis, enteric fistulas, hemorrhages.

There are three degrees of severity of AP:

Usually, mild AP account for 80–85% of cases, while severe AP is reported in 15–30% of patients [6].


*Edited by Paolo Saona Hoffmann*

This edited volume aims to discuss the most contemporary state of the determinants of the firm value. This book presents theoretical works as well as empirical studies that contrast the arguments offered by the leading, ground-breaking theories on the firm value.

What variables determine the firm value? Are these determinants controllable or uncontrollable by the managers of the companies? Is the impact of corporate governance systems on the firm value symmetrical between different institutional contexts? Do the financial reports affect the value of the firm? What role does corporate social responsibility play as a determinant of the firm value? These and other questions are analyzed and scrutinized step by step throughout this book.

Published in London, UK © 2018 IntechOpen © pinkomelet / iStock

Firm Value - Theory and Empirical Evidence

Firm Value

Theory and Empirical Evidence

*Edited by Paolo Saona Hoffmann*


**7. References**

Examples of fluid engineering and application of computer simulation. (Han zhan-zhong, 2005).

**18**

*Brazil*

*LabOceano – COPPE/UFRJ*

**A Three-Dimensional Numerical Simulation of**

Offshore oil and gas production is advancing fast towards water depth deeper and deeper. In the last couple of years, offshore oil production depth world records have been successively superseded. Oil industry is about to achieve production in fields approaching 2000m water depth while keeping on exploring new oil reservoirs in ocean depths close to 3000m. As water depth increases, the distance from oil field to mainland depots increases at similar rates and more hostile the ocean environment becomes. In such operational conditions the use of VLCC ship tanker, as a production unit, has been proved to be

Good hydrodynamic characteristics in severe sea environments, adequate storage capability and possibly the low prices of used tanker hulls in the ship market are the main reasons to justify the increasing popularity of tanker hull as production units (Floating Storage and Offloading – FSO and Floating Production Storage and Offloading – FPSO) among offshore oil producer companies. The complete assessment of the dynamic behavior of moored tankers depends very much on the accuracy of the hydrodynamic loading and response evaluation that need to be performed. Potential and viscous effects on the FSO/FPSO come into play equally important role on the acting flow around the ship hull. Furthermore, translational and rotational motions of the hull have to be incorporated all together into the

Recently, Computational Fluid Dynamics (CFD) has been experiencing rapid advances due to both computer technology progress and efficient algorithms that have been developed to solve the Navier-Stokes (N-S) equations used in the flow analysis around ship hulls. The present work is a contribution to the numerical solution of the viscous flow around ship-like bodies. In the present work, the slightly compressible Navier-Stokes equations (Wanderley et al. [1]) are solved through the conservative upwind TVD scheme of Roe [2] and Sweby [3]. This finite difference method is second order accurate in space and time. The physical domain is discretized using a Cartesian mesh and the no-slip boundary condition on the body surface is imposed through the Immersed Boundary Method (IBM), Marcelo et al. [4]. The Cartesian mesh is not conformed to the body contour and the IBM is used to inform the fluid flow the presence of a body through a force field added to the momentum equations. The code was implemented using the message passing interface (MPI) and can be run in a cluster with an arbitrary number of computers. Numerical results were obtained for the flows around a

**1. Introduction**

technically and economically appealing.

analysis to produce a realistic picture of the physical problem.

**the Free Surface Flow Around a Ship Hull**

J. B. V. Wanderley, M. Vitola, S. H. Sphaier and C. Levi