**Acknowledgements**

The Eva Crane Trust has partly funded DR research conducted in this chapter. This chapter was written with the kind assistance from Marisa Greco, Christina Belcev, Alex Belcev, Nell Greco, Pedro Velasco and Isabella Belcev.


*Edited by Parinya Sanguansat*

This book is aimed at raising awareness of researchers, scientists and engineers on the benefits of Principal Component Analysis (PCA) in data analysis. In this book, the reader will find the applications of PCA in fields such as image processing, biometric, face recognition and speech processing. It also includes the core concepts and the stateof-the-art methods in data analysis and feature extraction.

ISBN 978-953-51-0195-6

Principal Component Analysis

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**2.4 Osmotic flows in cryopreserving mammalian embryos**

#### **2.4.1 Slow-cooling**

In a general slow-cooling procedure, embryos are immersed into permeating cryoprotectants. Intracellular water leaves the cells by osmosis and re-enters the cells together with the permeating cryoprotectants by diffusion. A temporal osmotic equilibrium state is acquired at the end. A 1-2M permeating cryoprotecting agent(s) is often used in slow-cooling. To cryopreserve the embryos, embryos and the surrounding freezing medium are loaded into a plastic straw and subjected to cooling to a temperature slightly lower than the freezing point, i.e. at about -7°C. Controlled ice nucleation is initiated by touching the straw with a cooler surface (e.g., a pair of forceps) to initiate the growth of ice inside the straw. The embryos themselves remain unfrozen but supercooled. The removal of water from the solution by the growing ice crystals increases the solute concentration of the extracellular medium. By osmosis, the intercellular water leaves the cells, resulting in an increase of cryoprotectant concentration in the cells. The subsequent slow cooling further dehydrates the embryos and concentrates the cryoprotectant in the cells to promote intracellular solidification, without intracellular ice formation, at a sufficiently low temperature. If the cooling is too fast, it leads to intracellular ice formation because the intracellular solute has not yet achieved a sufficiently high concentration. Too slow a cooling rate causes cells' death due to the prolonged exposure to hypertonic conditions. The cooling rate must be carefully controlled for each embryonic stage of each species because the permeability of cell membranes, and thus the hydrodynamics, of different samples, can be different.

#### **2.4.2 Vitrification**

In cryopreservation by vitrification (or rapid-cooling), equilibration of cryoprotectants in the embryos and the cryoprotecting medium are not required. Embryos are first permeated by cryoprotecting agents at a low concentration and then immersed in a moderately high concentration (4M or above) of the same cryoprotecting agent, sometimes together with non-permeating cryoprotectants such as 0.5M sucrose. In the presence of the nonpermeating cryoprotectants, the embryos shrink osmotically, thus further increasing concentration of intracellular cryoprotectants. The high concentration of cryoprotecting agents in the medium prevents efflux of intracellular cryoprotecting agents by diffusion (Figure 4). The embryos are then loaded into a container and are rapidly cooled to solidify the embryos without the formation of ice crystals (Rall, 1987). Mathematical modelings such as the relativistic permeability approach is able to simulate the osmotic curve in these

embryos. When the mouse morulae were stored in 1.5M ethylene glycol or glycerol for 6 hours, the majority (>75%) of the embryos retained the capacity to develop into expanded blastocysts (Kasai et al., 1981). To minimize the toxic effect of the cryoprotecting solution to the embryos while retaining the cryoprotecting function, a mixture of two or more cryoprotecting agents could be used to decrease the relative concentration of each chemical. Macromolecules such as polyethylene glycol, ficoll and polyvinylpyrrolidone, which increase the viscosity of a solution, thus slowing down water molecules associating to form ice crystals when cooling, can also lower the concentration of cryoprotecting agents to be

In a general slow-cooling procedure, embryos are immersed into permeating cryoprotectants. Intracellular water leaves the cells by osmosis and re-enters the cells together with the permeating cryoprotectants by diffusion. A temporal osmotic equilibrium state is acquired at the end. A 1-2M permeating cryoprotecting agent(s) is often used in slow-cooling. To cryopreserve the embryos, embryos and the surrounding freezing medium are loaded into a plastic straw and subjected to cooling to a temperature slightly lower than the freezing point, i.e. at about -7°C. Controlled ice nucleation is initiated by touching the straw with a cooler surface (e.g., a pair of forceps) to initiate the growth of ice inside the straw. The embryos themselves remain unfrozen but supercooled. The removal of water from the solution by the growing ice crystals increases the solute concentration of the extracellular medium. By osmosis, the intercellular water leaves the cells, resulting in an increase of cryoprotectant concentration in the cells. The subsequent slow cooling further dehydrates the embryos and concentrates the cryoprotectant in the cells to promote intracellular solidification, without intracellular ice formation, at a sufficiently low temperature. If the cooling is too fast, it leads to intracellular ice formation because the intracellular solute has not yet achieved a sufficiently high concentration. Too slow a cooling rate causes cells' death due to the prolonged exposure to hypertonic conditions. The cooling rate must be carefully controlled for each embryonic stage of each species because the permeability of cell

membranes, and thus the hydrodynamics, of different samples, can be different.

In cryopreservation by vitrification (or rapid-cooling), equilibration of cryoprotectants in the embryos and the cryoprotecting medium are not required. Embryos are first permeated by cryoprotecting agents at a low concentration and then immersed in a moderately high concentration (4M or above) of the same cryoprotecting agent, sometimes together with non-permeating cryoprotectants such as 0.5M sucrose. In the presence of the nonpermeating cryoprotectants, the embryos shrink osmotically, thus further increasing concentration of intracellular cryoprotectants. The high concentration of cryoprotecting agents in the medium prevents efflux of intracellular cryoprotecting agents by diffusion (Figure 4). The embryos are then loaded into a container and are rapidly cooled to solidify the embryos without the formation of ice crystals (Rall, 1987). Mathematical modelings such as the relativistic permeability approach is able to simulate the osmotic curve in these

**2.4 Osmotic flows in cryopreserving mammalian embryos**

used in vitrification.

**2.4.1 Slow-cooling**

**2.4.2 Vitrification**

processes to facilitate the optimization of vitrification protocols in future (Katkov and Pogorelov, 2007).


*3.4. Optical Observation of Cracks*

The prisms produced for this research were pre-cracked after 28 days of curing under the two regimes. Representative images of optical observation under the curing regimes are given in Figure 7. The images were captured when the cracks were formed and correspond to 0 days. The images captured at 14 and 28 days after cracking correspond to 28 + 14 and 28 + 28 days total age. Table 6 shows representative measurements of the crack width evolution during the curing.

The images of neat cement depict the autogenous self-healing that the system could perform under these specific conditions. The width decrease is not significant (Table 6) in both curing conditions. At 14 days, there are open parts in the cracks that have not healed, while at 28 days, the situation is improved. However, unhealed areas still remain.

The water immersion promoted the formation of crystals in NC-cement pastes and allowed the healing of up to 14 days. The mean crack width was 0.156 mm at 0 days and is healed, as depicted in Figure 7. The newly formed material also overflowed on the surface of very small cracks. The 28 days captions showed a progression of this crystallization. A greater quantity of the material was observed on the surface of the cracks and in a broader area around them. The system presented healing to a smaller degree when it was subjected to wetting-drying cycles. The crack width decreased significantly up to 14 days, from 0.106 mm to 0.031 mm, and it was eliminated at 28 days.

The combination of nanoparticles showed efflorescing phenomena after 14 days of cracking, but the cracks were not healed at that age. Healing was recorded up to 28 days for samples cured in the water, and a satisfactory crack closure was recorded for samples kept in the wetting-drying regime.

**Table 5.** EDS analysis of the micrographs shown in Figure 6.

**Figure 7.** *Cont.*

**Figure 7.** *Cont.*

**Figure 7.** Optical microscopy observation of the cement pastes at 28 + 0 days of the cracking, at 28 + 14 days and 28 + 28 days, cured in water immersion (w) and wetting-drying cycles (c) (Scale equals to 1 mm).

**Table 6.** Crack width evolution (mean values of 14 measurements).


**5.1 Origin and epidemiology of high-grade serous ovarian cancer**

EOC genomic predisposition is now recognized in about 15% of affected women, where Breast cancer susceptibility genes *BRCA1* and *BRCA2* were identified as the main causative agents of hereditary EOC. Different forms of mutations in these genes and other double-strand DNA break repair genes are mainly associated with susceptibility to HGSOC [1].

High-grade serous ovarian cancer (HGSOC) is the most common form of EOC, accounting for about 75% of all EOC (**Figure 1**). It has been found to originate from the fallopian tube epithelium due to its link with IGF-1R/AKT pathway, which is activated by follicular fluid [17]. However, the molecular basis of how it is transferred to the ovaries is yet to be understood. A recent study revealed that follicular fluid plays a vital role in events leading to the development and intraperitoneal metastasis of HGCOS, by supporting migration, proliferation, invasion, anchorage, adhesion and anoikis insensitivity [18].


**Acknowledgements**

*Water Chemistry*

the stretching of P-O (H) in the HPO**−**2

and the peaks observed at 665 and 1651 cm**−**1

*Infrared spectrum of pure nCDPD doped with Ni, Co, and Cu.*

signals obtained at 3548, 3484, 3278, and 3166 cm**−**1

the water molecule in the apatite. Finally, a band at 2944 cm**−**1

1138, 1120, 1065, and 1004 cm**−**1

these signals are related to the PO3

1215 cm**−**1

**Figure 14.**

metals directly.

**4. Conclusion**

4. On the other hand, the signals found at

are related to the binding vibrations

are related to the extension of

was also observed,

are attributed to the stretching of the P-O link. At

**−**4 and OH groups, with which it can be inferred

the signal generated by the flexion of the O-H group plane is presented,

and physical vibrations of the water molecule. Additionally, a shoulder at 1725 cm**−**1 was observed, which is related to the flexural stress of the water molecule, and the

directly related to the stretching of PO-H that occurs in HPO [1, 41, 42]. In the case of the nDCPD spectra with the different metal ions, it was observed that several peaks decrease in intensity or disappear as a result of the presence of Ni, Co, and Cu;

that the groups present on the surface of this apatite participate in the adsorption of

Based on the thermodynamic parameters obtained at different temperatures and concentrations of brushite (nDCPD), used as an adsorbent material in the removal of different heavy metals (Ni, Co, and Cu), from aqueous solutions, it can be concluded that the adsorption of heavy metals with nDCPD is feasible and that the adsorption capacity of nDCPD increases at a higher temperature. The percentages of removal achieved show that nDCPD has a higher affinity for adsorbing Cu and Co compared to Ni. From the data obtained from the adsorption isotherms, it can be inferred that the process of removing the metal ions in solution on the surface of nDCPD is carried out, forming a heterogeneous monolayer since the model that best fits to the data obtained in the SIPS is a combination of the Langmuir and Freundlich models. The experimental data obtained also show that there are different models to describe the kinetic process of adsorption, such as the pseudo-firstorder model, which suggests that only one surface site is needed for adsorption to take place, as well as the pseudo-second-order model which indicates that two sites are needed for each ion molecule and that each site requires different adsorption

**106**

The authors thank the Interdisciplinary Professional Unit of Engineering Campus Guanajuato of the IPN, the Center for Nanoscience and Nanotechnology (CNyN) of the UNAM, and the Colombian Polytechnic "Jaime Isaza Cadavid" for the infrastructure provided for the realization of this project and the Research and Postgraduate Secretary for the financial support for this work (SIP: 20170577) and to the BIOCATEM Network.


**1. Introduction**

The ferroelectric materials are very important for technological applications in general, like sensor and actuators they are important part of electronic devices. The temperature, pressure, electric and electromagnetic field nonlinear response of ferroelectrics make them ideals like active elements due to pyroelectric and piezoelectric effects [1–4]. Ferroelectrics are used like ultrasonic generator; high voltage transforms and accelerators [1–4], also, they are using in optical components, piezoelectric transducers, and pyroelectric sensors [1–4]. By fabrication ferroelectric materials born with electric dipole different to zero, *P* 6¼ 0; this electric property is by asymmetry crystalline center structure. The **Figure 1** shows two crystalline structures, first is a symmetry perovskite structure with *P* ¼ 0, and second is an asymmetry perovskite structure with *P* 6¼ 0 typical to lead titanate (*PbTiO*3) [5]. The lead titanate (*PbTiO*3) is used intensively in electronic devices [5–7]. The *PbTiO*3 ceramic had perovskite structure (tetragonal structure) with ratio *c=a* ¼ 1*:*064, where *c* ¼ 4*:*154Å, *a* ¼ 3*:*899Å, the **Figure 1a** shows the lead ion ð Þ *Pb* occupies places A, the titanium ion (Ti) occupies the places B and the oxygen ion is on the parallelepiped faces occupies the places O [8].

**Figure 1.**

*The perovskite structure for PbTiO*3 *a) Centre symmetry and b) non Centre with ratio c=a* ¼ 1*:*064*, where c* ¼ 4*:*154̊A*, a* ¼ 3*:*899̊A*.*

The ceramic fragility is reduced for inserting rare earths or transitions metal atoms [6–8]. The partial or total substitution of Pb or Ti produces modified compounds with new electrical polarization and modifies their structure depending of doping atoms and the interactions material [8]. The changes in the structure could be small or big depending to the kind of dopand and the percentage of them [1–8]. If the dopand is a paramagnetic ion it could be detected by paramagnetic resonance (EPR) and it is capable to sense structure changes principally by spin-orbit interactions [9–11].

The paramagnetic resonance technique (EPR) detects the spin-orbital magnetic interaction in doped ferroelectrics when the dopant is a rare earth with a paramagnetic ion. If dopant is chromium (Cr), it can give a paramagnetic state, in this case the electron paramagnetic resonance EPR technique is capable to detect paramagnetic ions, and the EPR-technique is high sensitivity to spin interactions due crystalline structure, nuclear interaction (hyperfine), or anisotropies (orientations) [9–11]. The nondestructive EPR-technique is applied to organic and inorganic molecules, ions, and atoms that have unpaired electrons that have information about oxidation state and spin state due to electron unpaired spin [9–11]. The EPR technique use microwave energy to induce resonances or electron transitions between electronic energy levels separated by applying magnetic field for paramagnetic compounds or substances. The EPR typical spectra for Cr, Fe, Mn, Co, Ni, Cu, V, etc. [11–15] contained information about the *g*-factor, the *A*-hyperfine couple and crystalline field factors, *D* and *E*.

#### **2. Atoms into magnetic field** *H* !

The energy interaction between magnetic moment *μ* ! and extern magnetic field *H* ! is given by *W* ¼ *μ* ! ∙ *H* ! . The electronic orbital movement is the cause of atomic momentum for the atom *μ* !, it is proportional to angular momentum *L* ! given by *μ* ! *L* ¼ *e* 2*mc L* ! , where *L* ! is the orbital angular momentum, *e* is the electron charge, *m* is the electron mass, and *c* is the speed of light. The gyromagnetic ratio is defined by *γ e* 2*mc* ¼ 1*:*7 107 *rad gauss*-1 , so the atomic momentum is given by *μ* ! *L* ¼ *γL* ! [9].

Otherwise, the intrinsic magnetic momentum for electron is *μ* ! *S* ¼ 2 *e* 2*mc S* ! , where *S* ! is the spin vector associated to electron spin. The total magnetic moment is given by the spin and orbital momentum addition *μ* ! ¼ *μ* ! *L* þ *μ* ! *S*. If the total magnetic moment is placed into magnetic field *H* ! , the total magnetic energy is given *Paramagnetic Transitions Ions as Structural Modifiers in Ferroelectrics DOI: http://dx.doi.org/10.5772/intechopen.95983*

by *W* ¼ *μ* ! ∙ *H* ! ¼ *μH* cos *θ*, where *θ* is the angle between vectors *μ* ! and *H* ! , the angle could varies continually in the classical description, however, in the quantum mechanics description the variation is quantized with 2*J* þ 1 orientations, where *J* is the quantum number for the total angular momentum given by ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi *J J*ð Þ þ 1 p ℏ [9, 12]. The allowed projections *J*, when the system is quantized along the magnetic field direction, are given by *m j*ℏ, where *m j* is the quantum magnetic number with values from *J*, *J* - 1, … , -*J* [15].

The simplest case is just only the spin electronic momentum (atoms in base state 2 *S*1*=*2) with *mS* ¼ *S*, *S* - 1, … , -*S*, with *S* the total electronic spin, and the projections are *μ* ! *S* ¼ 2 *eh* 4*πmc mS* where for *S* ! operator the eigenvalue *mS*ℏ was substituted. The quantity *eh* 4*πmc βe* ¼ 9*:*2741 10-21*erg=gauss* is called Bohr's magneton, then *μ* ! *S* ¼ *βemS* and the energy values allowed for the atom placed into a magnetic field *H* ! , are *EmS* ¼ *μSH* ¼ 2*βemSH* (Zeeman's energy). For an electron isolate quantum electrodynamic correction is necessary [9–15] replacing the number 2 for the *ge* ¼ 2*:*00023. In the case for isolate spin *S* ¼ 1*=*2, the 2*S* þ 1 energy levels are *geβeH* equally spaced, the **Figure 2** shows the case for *S* ¼ 1*=*2 [9–15].

For degenerated orbital state (*L* ¼6 0) where exist and important L and S coupling (Russell-Saunders coupled) [9–15] there are *J* ¼ *L* þ *S*, *L* þ *S* - 1,…, ∣*L* - *S*∣, and for each *J* there are *mJ* ¼ *J*, *J* - 1,…, -*J* values, so the energy for each degenerated state is *EmJ* ¼ *gJ βemJH*, where *gJ* ¼ 1 þ *J J*ð Þþ þ1 *S S*ð Þ þ1 *L L*ð Þ þ1 2*J J*ð Þ þ1 is the splitting Landé factor. For example, the state 2 *P*1*=*2 have *S* ¼ 1*=*2, *L* ¼ 1, *J* ¼ 1*=*2 and then *g* ¼ 3*=*2. Of course, when *L* ¼ 0 the *gJ* ¼ *ge* [9].

#### **Figure 2.**

*EPR scheme for system with S = 1/2. The Zeeman's effect for S* ¼ 1*=*2 *split the degenerate state energy into W*1 *and W*2 *and the microwave photon hν provide the energy for the transition between them [14].*


*Edited by Desiree Nedra Karunaratne and Geethi Pamunuwa*

Food additives is intended to provide the readers with knowledge on some very significant aspects of the food additives currently in use. Food additives have become essential in the food sector with the rising need for food processing and preservation. However, the use of food additives is regulated imposing strict rules as the impact of those additives on health cannot be neglected. The first chapter starts off with a general overview of food additives highlighting the novel trends that enhance the attributes of those additives. Thereafter, the chapters are devoted mainly to plantderived food additives and microbially derived food additives. The main topics discussed under 'additives from plant origin' are the efficacy of beetroot formulations as a source of nitrate ions, plant-derived food preservatives and plant-derived food additives used in meat and meat-based products. The further chapters discuss 'additives from microbial origin' focusing on lactic acid bacteria and additives derived from lactic acid bacteria and food additives used in 'bread-making'. Overall, this manuscript emphasises the concept of 'clean labelling' and the importance of natural food additives.

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## Food Additives

*Edited by Desiree Nedra Karunaratne and Geethi Pamunuwa*


**3. Maternal and umbilical cord blood modifications**

PE is a disorder involving maternal and placental changes. It involves fetal complications, such as, prematurity and IUGR, which are the most representative. Newborns, whose mothers develop PE, usually present a lower birth weight than infants born from mothers with a normal pregnancy (Catarino et al., 2008a). Moreover, newborns small for gestational age, as well as newborns with Apgar score below 7, are frequently observed in PE (Catarino et al., 2008a).

There are several studies concerning maternal modifications in PE, but there are few studies carried out in umbilical cord blood. We will address the effect of some modifications usually observed in PEc women, such as, in lipid profile, in hematologic profile, inflammatory and antioxidant markers, angiogenic/anti-angiogenic factors and hemostatic disturbances, in umbilical cord blood.

#### **3.1 Angiogenic/anti-angiogenic factors**

A normal placental development is essential for adequate feto-maternal nutrients and gas exchanges. In addition, placenta is an important endocrine organ that synthesizes various hormones, cytokines and angiogenic growth factors. These factors are released into the maternal circulation, and may contribute to changes in endothelial function. It is

factors. It has been shown that PEc placentas present an increased expression of sFlt1 (Gu et al., 2008), and a decreased expression of PlGF (Gu et al., 2008). These placental factors and cytokines appear to be released in maternal circulation, resulting in a generalized endothelial dysfunction, causing the multisystem complications of a PEc pregnancy

(Rusterholz et al., 2007; Maynard et al., 2008) (Fig. 1).

Fig. 1. Possible mechanisms involved in the pathogenesis of preeclampsia

PE is a disorder involving maternal and placental changes. It involves fetal complications, such as, prematurity and IUGR, which are the most representative. Newborns, whose mothers develop PE, usually present a lower birth weight than infants born from mothers with a normal pregnancy (Catarino et al., 2008a). Moreover, newborns small for gestational age, as well as newborns with Apgar score below 7, are frequently observed in PE (Catarino

There are several studies concerning maternal modifications in PE, but there are few studies carried out in umbilical cord blood. We will address the effect of some modifications usually observed in PEc women, such as, in lipid profile, in hematologic profile, inflammatory and antioxidant markers, angiogenic/anti-angiogenic factors and hemostatic disturbances, in

A normal placental development is essential for adequate feto-maternal nutrients and gas exchanges. In addition, placenta is an important endocrine organ that synthesizes various hormones, cytokines and angiogenic growth factors. These factors are released into the maternal circulation, and may contribute to changes in endothelial function. It is

**3. Maternal and umbilical cord blood modifications**

et al., 2008a).

umbilical cord blood.

**3.1 Angiogenic/anti-angiogenic factors**

recognized that placenta has an important role in the protection and development of the fetus, promoting feto-maternal exchanges which are essential to a normal pregnancy. On the other hand, it is also recognized that in PE there are changes in placental development (Pijnenborg et al., 2006; Young et al., 2010), which can compromise the feto-maternal exchange, limiting fetal development, and trigger a maternal and fetal response to adapt to these changes.

Angiogenic factors have been subject of intensive research in recent years, as they appear to be involved in the aetiology of PE. Several studies show a decrease in the concentration of PlGF (Polliotti et al., 2003; Levine et al., 2004) and an increase in circulating levels of sVEGFR-1 (McKeeman et al., 2004; Levine et al., 2004) in PEc women. Nevertheless, there is some controversy regarding the concentration of VEGF in pregnant women with PE (Simmons et al., 2000; McKeeman et al., 2004). Since sVEGFR-1 is an antagonist of PlGF and VEGF, there is a reduction of the effects of these factors, i.e., a change in angiogenesis and in endothelial function (Luttun & Carmeliet, 2003). The anti-angiogenic effect in pregnant women with PE seems to disappear after delivery (Maynard et al., 2008; Myatt & Webster, 2009), strengthening the involvement of placental factors in PE. VEGF plays an important role in vascular development, especially at placenta level, but also induces NO and prostaglandins synthesis, which are mediators of vasodilation (Myatt & Webster, 2009).

The processes of vasculogenesis/angiogenesis are crucial for the development of uteroplacental circulation and placenta. In a study performed by our group (Catarino et al., 2009a), we observed a disturbance of the angiogenic/anti-angiogenic factors in the maternal circulation in PE. PEc women had significantly higher levels of sVEGFR-1 (Fig. 2B) and VEGF values and significantly lower PlGF levels (Fig. 2A). This disruption in angiogenesis factors seems to be associated with placental dysfunction, since these factors are mainly produced by the placenta (Kaufmann et al., 2004), during pregnancy.

We also observed a positive correlation between levels of PlGF and maternal placental weight, in PEc pregnancy, which highlights the importance of PlGF in placental development (Catarino et al., 2009a). It is worthy to note that sVEGFR-1, an inhibitor of PlGF and VEGF, was significantly correlated with the amount of proteinuria, a marker of PE severity, suggesting an important role of sVEGFR-1 in the pathogenesis of PE (Catarino et al., 2009a). The abnormal development of the placenta in PE, reduces the placenta perfusion and may also contribute to the observed increase in the amounts of sVEGFR-1 and VEGF, since the tissue hypoxia regulates its production, stimulating it.

In cord blood samples of PEc cases, we observed a significant decrease in PlGF (Fig. 2C) and VEGF concentrations and a significant increase in the levels of sVEGFR-1 (Fig. 2D) (Catarino et al., 2009a). This disruption, particularly at values of PlGF and sVEGFR-1, seems to be an indirect indicator of the involvement of placental dysfunction in PE, since both are produced primarily in the placenta.

The observed correlation between mother and child for sVEGFR-1 seems to indicate that the release of these factors by placenta, occurs both for the maternal and fetal circulation. However, the levels of sVEGFR-1 are higher in the blood of mothers, when compared with cord blood levels, which allows us to suppose that decidua, since it is able to secrete sVEGFR-1 (Lockwood et al., 2007), may also contribute for maternal levels of sVEGFR-1.

Umbilical Cord Blood Changes in Neonates from a Preeclamptic Pregnancy 277

The hemostatic system is altered in PE, which may increase the incidence of maternal and fetal complications. In PEc women, a decrease in the number of platelets compared with normal pregnancy is frequently described (Howarth et al., 1999; Osmanağaoğlu et al., 2005), and thrombocytopenia may occur. Maternal thrombocytopenia is usually associated with more severe pathology, including the HELLP syndrome. Harlow et al. (2002) stated that the decrease in the platelets value is the result of an increase in consumption, as they observed intense platelet activation in patients with PE compared with other pregnancy hypertensive diseases and normal pregnancy. On the other hand, the increase in the mean platelet volume (increased younger platelets, with larger size) (Howarth et al., 1999) and the higher thrombopoietin circulating levels (specific growth factor that promotes thrombocytopoiesis) in women with PE (Johnson et al., 2001), reflect an increase in platelet production. Thus, the

The fibrinolytic system is usually disturbed in PE, due to increased plasminogen activator inhibitor (PAI)-1 and PAI-2. Several studies describe a significant increase in plasma levels of PAI-1 in cases of PE compared to normal pregnancy (Belo et al., 2002a; Tanjung et al., 2005; Sartori et al., 2008; Hunt et al., 2009). The expression of PAI-1 is also elevated in placentas from PEc pregnant women, and PAI-1 plasma levels seem to be positively correlated with the severity of placental damage (Estelles et al., 1998). PAI-2 can be considered a marker of placental function, as it is mainly synthesized at trophoblastic tissue. In PE a decrease in plasma PAI-2 seems to occur (Roes et al., 2002; Tanjung et al., 2005; Sartori et al., 2008), suggesting a modification of placental function. Considering the increased PAI-1 and the decrease of PAI-2, some authors suggest that the rise in the ratio PAI-1/PAI-2 in maternal plasma may be considered a marker of PE (Chappell et al., 2002; Hunt et al., 2009). Several authors report that tissue plasminogen activator (tPA), one of the physiological plasminogen activators, is increased in PE (Belo et al., 2002a; Tanjung et al., 2005; Hunt et al., 2009). Increased levels of D-dimers (fragments of fibrin degradation products) were also reported in PE (Belo et al., 2002a; Hunt et al., 2009), reflecting an increase in the activation process of coagulation and fibrinolysis. The production of Ddimer depends on the formation of thrombin, resulting from activation of the coagulation and fibrinolytic systems. At hemostatic level changes in other parameters, such as a decrease in antithrombin III (Osmanağaoğlu et al., 2005; Tanjung et al., 2005) and an increase in thrombin-antithrombin complex (TAT) in PE (Hunt et al., 2009) were

In order to clarify the involvement of hemostatic abnormalities in PE, we assessed fibrinolytic markers, in particular, tPA and PAI antigens and fibrin degradation products (D-dimers). We found that the values of tPA (Fig. 3A) and PAI-1 were significantly higher in PE, without changes in D-dimers (Catarino et al., 2008b). The high levels of tPA and PAI-1 suggest endothelial dysfunction in this syndrome, as both substances are produced by the endothelial cell and exert antagonic roles in the fibrinolytic process. In addition, both PAI-1 and tPA present positive correlations with proteinuria, suggesting that the severity of PE is associated with increased activation/endothelial dysfunction (Catarino et al., 2008b). Furthermore, the maternal endothelial (dys)function, appears to be related to placental (dys)function, considering the positive correlation that we observed between tPA

and sVEGFR-1 values in normal and PEc pregnancy (Catarino et al., 2009a).

reduction of platelet count in PE seems to be due to a higher consumption.

**3.3 Hemostasis**

described.

Fig. 2. Concentrations of PlGF in maternal blood (A) and cord blood (C), and sVEGFR-1 levels in maternal blood (B) and cord blood (D) from women with preeclampsia (PE) or with normal pregnancy (N). (Adapted from Catarino et al., 2009a)

#### **3.2 Endothelial dysfunction**

Several investigators support the hypothesis that in PE endothelial dysfunction occurs, what contributes to the onset of maternal clinical manifestations. Furthermore, this dysfunction seems to be the link between changes in placental and multisystem complications (Roberts & Gammil, 2005; Young et al., 2010).

In literature, a large number of studies describing changes in endothelial function markers in PE can be found, in particular, an increase of plasma endothelin-1 (Baksu et al., 2005a; Bernardi et al., 2008a), soluble vascular cell adhesion molecule (sVCAM), plasma fibronectin (Aydin et al., 2006; Dane et al., 2009) and tissue fibronectin (Powers et al., 2008), a decrease of plasma and urinary nitric oxide (NO) levels (Baksu et al., 2005a; Mao et al., 2009).

PE is thus associated with a decrease in vasodilatory mediators such as NO and prostacyclin, and an increase in vasoconstrictive mediators such as endothelin-1, angiotensin II and thromboxane A2 (Myatt & Webster, 2009). There are also markers related with hemostasis that are altered in PE and can also be considered as markers of endothelial dysfunction (see Hemostasis section). as

In PE, there are several entities that may induce or contribute to injury and/or endothelial dysfunction. These include changes at the level of angiogenic factors/anti-angiogenic, the presence of oxidative stress, exacerbation of the inflammatory process, changes in lipid profile and also a hypoxic environment (Myatt & Webster, 2009).

#### **3.3 Hemostasis**

276 From Preconception to Postpartum

Fig. 2. Concentrations of PlGF in maternal blood (A) and cord blood (C), and sVEGFR-1 levels in maternal blood (B) and cord blood (D) from women with preeclampsia (PE) or with

Several investigators support the hypothesis that in PE endothelial dysfunction occurs, what contributes to the onset of maternal clinical manifestations. Furthermore, this dysfunction seems to be the link between changes in placental and multisystem complications (Roberts

In literature, a large number of studies describing changes in endothelial function markers in PE can be found, in particular, an increase of plasma endothelin-1 (Baksu et al., 2005a; Bernardi et al., 2008a), soluble vascular cell adhesion molecule (sVCAM), plasma fibronectin (Aydin et al., 2006; Dane et al., 2009) and tissue fibronectin (Powers et al., 2008), a decrease

PE is thus associated with a decrease in vasodilatory mediators such as NO and prostacyclin, and an increase in vasoconstrictive mediators such as endothelin-1, angiotensin II and thromboxane A2 (Myatt & Webster, 2009). There are also markers related with hemostasis that are altered in PE and can also be considered as markers of endothelial

In PE, there are several entities that may induce or contribute to injury and/or endothelial dysfunction. These include changes at the level of angiogenic factors/anti-angiogenic, the presence of oxidative stress, exacerbation of the inflammatory process, changes in lipid

factors/anti-angiogenic,

of plasma and urinary nitric oxide (NO) levels (Baksu et al., 2005a; Mao et al., 2009).

normal pregnancy (N). (Adapted from Catarino et al., 2009a)

profile and also a hypoxic environment (Myatt & Webster, 2009).

**3.2 Endothelial dysfunction**

& Gammil, 2005; Young et al., 2010).

dysfunction (see Hemostasis section).

The hemostatic system is altered in PE, which may increase the incidence of maternal and fetal complications. In PEc women, a decrease in the number of platelets compared with normal pregnancy is frequently described (Howarth et al., 1999; Osmanağaoğlu et al., 2005), and thrombocytopenia may occur. Maternal thrombocytopenia is usually associated with more severe pathology, including the HELLP syndrome. Harlow et al. (2002) stated that the decrease in the platelets value is the result of an increase in consumption, as they observed intense platelet activation in patients with PE compared with other pregnancy hypertensive diseases and normal pregnancy. On the other hand, the increase in the mean platelet volume (increased younger platelets, with larger size) (Howarth et al., 1999) and the higher thrombopoietin circulating levels (specific growth factor that promotes thrombocytopoiesis) in women with PE (Johnson et al., 2001), reflect an increase in platelet production. Thus, the reduction of platelet count in PE seems to be due to a higher consumption.

The fibrinolytic system is usually disturbed in PE, due to increased plasminogen activator inhibitor (PAI)-1 and PAI-2. Several studies describe a significant increase in plasma levels of PAI-1 in cases of PE compared to normal pregnancy (Belo et al., 2002a; Tanjung et al., 2005; Sartori et al., 2008; Hunt et al., 2009). The expression of PAI-1 is also elevated in placentas from PEc pregnant women, and PAI-1 plasma levels seem to be positively correlated with the severity of placental damage (Estelles et al., 1998). PAI-2 can be considered a marker of placental function, as it is mainly synthesized at trophoblastic tissue. In PE a decrease in plasma PAI-2 seems to occur (Roes et al., 2002; Tanjung et al., 2005; Sartori et al., 2008), suggesting a modification of placental function. Considering the increased PAI-1 and the decrease of PAI-2, some authors suggest that the rise in the ratio PAI-1/PAI-2 in maternal plasma may be considered a marker of PE (Chappell et al., 2002; Hunt et al., 2009). Several authors report that tissue plasminogen activator (tPA), one of the physiological plasminogen activators, is increased in PE (Belo et al., 2002a; Tanjung et al., 2005; Hunt et al., 2009). Increased levels of D-dimers (fragments of fibrin degradation products) were also reported in PE (Belo et al., 2002a; Hunt et al., 2009), reflecting an increase in the activation process of coagulation and fibrinolysis. The production of Ddimer depends on the formation of thrombin, resulting from activation of the coagulation and fibrinolytic systems. At hemostatic level changes in other parameters, such as a decrease in antithrombin III (Osmanağaoğlu et al., 2005; Tanjung et al., 2005) and an increase in thrombin-antithrombin complex (TAT) in PE (Hunt et al., 2009) were described.

In order to clarify the involvement of hemostatic abnormalities in PE, we assessed fibrinolytic markers, in particular, tPA and PAI antigens and fibrin degradation products (D-dimers). We found that the values of tPA (Fig. 3A) and PAI-1 were significantly higher in PE, without changes in D-dimers (Catarino et al., 2008b). The high levels of tPA and PAI-1 suggest endothelial dysfunction in this syndrome, as both substances are produced by the endothelial cell and exert antagonic roles in the fibrinolytic process. In addition, both PAI-1 and tPA present positive correlations with proteinuria, suggesting that the severity of PE is associated with increased activation/endothelial dysfunction (Catarino et al., 2008b). Furthermore, the maternal endothelial (dys)function, appears to be related to placental (dys)function, considering the positive correlation that we observed between tPA and sVEGFR-1 values in normal and PEc pregnancy (Catarino et al., 2009a).

Umbilical Cord Blood Changes in Neonates from a Preeclamptic Pregnancy 279

increase of lipid peroxidation (MDA), followed by decreased total antioxidant capacity and ascorbic acid levels in newborns whose mothers developed PE (Mehendale et al., 2008; Howlader et al., 2009), while other studies report no difference in lipid peroxidation

Experimental studies (Poston et al., 2006; Rahimi et al., 2009; Xu et al., 2010), performed in pregnant women at risk, showed no beneficial effect on the antioxidant consumption such as vitamins E and C, in preventing the development of PE, despite the evidence for the involvement of a state of oxidative stress in PE. In fact, according to these trials, antioxidant therapy does not appear to be sufficient to prevent the development of the PE, further increasing the risk of neonatal complications, including increased rate of newborns with low

There are various mechanisms by which oxidative stress is linked to the inflammatory process. During the inflammatory response production and release of reactive oxygen metabolites occurs leading to oxidative stress. On the other hand, products of oxidative stress, such as those resulting from lipid peroxidation, are considered pro-inflammatory.

PE may be considered as an exacerbated inflammatory condition compared with physiological pregnancy, which appears to contribute to endothelial dysfunction. Initially, there is a localized inflammatory response within the placenta, while in a second phase predominates a systemic inflammatory response. Numerous studies have reported an increase of pro-inflammatory cytokines such as IL-6 and TNF-α (Bernardi et al., 2008b; Guven et al., 2009; Ouyang et al., 2009) in pregnant women with PE, when compared with

Concerning acute phase proteins, C-reactive protein (CRP) is probably the most studied one, mainly due to its sensibility in detecting inflammation, with a significant increase being observed in PEc pregnancy (Belo et al., 2003; Tjoa et al., 2003; Guven et al., 2009). Tjoa et al. reported an increase in plasma concentration of CRP between 10 and 14 weeks of gestation in pregnant women who subsequently developed PE and gave birth to newborns with

Cell adhesion molecules (CAM), necessary for the adhesion of leukocytes to vascular endothelium, are also altered in PE. Despite some contradictory results, plasma levels of (sICAM)-1, soluble vascular cell adhesion molecule (sVCAM)-1, soluble platelet endothelial cell adhesion molecule (sPECAM)-1 and soluble E-selectin are raised in PEc pregnant

The neutrophil activation also seems to be associated with PE, as some studies mentioned an increase in circulating levels of myeloperoxidase (Mellembakken et al., 2001; Gandley et al., 2008) and elastase (Belo et al., 2003; Gupta et al., 2006), both released during the

The increase in inflammatory markers in the maternal circulation could result from the release of substances from the placenta (local inflammation), then triggering a systemically inflammatory response. Some authors have suggested that tissue hypoxia resulting from

(Tastekin et al., 2005; Braekke et al., 2006).

**3.5 Inflammation**

normotensive pregnant women.

growth restriction (Tjoa et al., 2003).

women (Kim et al., 2004; Chavarrνa et al., 2008).

degranulation of neutrophils in the inflammatory process.

birth weight (Poston et al., 2006; Rahimi et al., 2009).

As already mentioned, different hemostatic modifications are recognized in normal and in PEc pregnancy; however, the exact pattern of these changes in the fetus is still poorly understood. Some authors have reported a decrease in fibrinogen levels, but there were no differences in tPA, PAI-1 and D-dimer (Zanardo et al., 2005), while others reported an increase in PAI-1, however with no differences in tPA values (Roes et al., 2002). Higgins et al. (2000) suggest that infants are somehow protected, at hemostatic system level, since no differences in D-dimers were observed in newborns whose mothers developed PE (Higgins et al., 2000). In a study performed by our group (Catarino et al., 2008b), we also observed similar values of D-dimers. However, and similarly to what we observed in PEc mothers, significantly increased tPA values were found in the newborns of these women (Fig. 3B). Our results suggest that these changes do not arise in response to activation of coagulation, but as a result of endothelial cell dysfunction. Furthermore, we observed a relationship between placental dysfunction and endothelial dysfunction in fetal circulation in PE, suggested by the positive significant correlation that we identified between the levels of tPA and sVEGFR-1 in umbilical cord blood (Catarino et al., 2009a).

Fig. 3. Maternal (A) and fetal (B) tPA levels in PE and normal pregnancy. (Adapted from Catarino et al., 2008b)

#### **3.4 Oxidative stress**

In PE there is evidence of oxidative stress that results from an increased production of oxidizing agents that is not counteracted by antioxidant activity. In fact, an increase in reactive oxygen species, namely in superoxide anion, and a decrease in superoxide dismutase (SOD) activity is observed in trophoblast cells from PEc women (Wang & Walsh, 2001). Different studies demonstrate the development of oxidative stress in PE (Raijmakers et al., 2004; Roberts & Gammil, 2005); either by decreased concentrations of antioxidants, or indirectly by increased lipid peroxidation in maternal circulation, that results from the action of oxygen metabolites, provided by leukocyte activation and/or increased cellular metabolism. The oxidative stress can also take place at the placenta, as a result of the hypoxia/reoxygenation mechanism (intermittent placental perfusion) observed in placentas of pregnant women with PE (Hung & Burton, 2006).

The preterm infants have a lower antioxidant capacity and are therefore more susceptible to oxidative stress triggered at birth, which appears to be associated with complications, including retinopathy and bronchopulmonary dysplasia (Saugstad, 2003). However, there are conflicting results considering oxidative stress in newborns. Some studies describe an increase of lipid peroxidation (MDA), followed by decreased total antioxidant capacity and ascorbic acid levels in newborns whose mothers developed PE (Mehendale et al., 2008; Howlader et al., 2009), while other studies report no difference in lipid peroxidation (Tastekin et al., 2005; Braekke et al., 2006).

Experimental studies (Poston et al., 2006; Rahimi et al., 2009; Xu et al., 2010), performed in pregnant women at risk, showed no beneficial effect on the antioxidant consumption such as vitamins E and C, in preventing the development of PE, despite the evidence for the involvement of a state of oxidative stress in PE. In fact, according to these trials, antioxidant therapy does not appear to be sufficient to prevent the development of the PE, further increasing the risk of neonatal complications, including increased rate of newborns with low birth weight (Poston et al., 2006; Rahimi et al., 2009).


**1. Introduction**

Orthopaedic procedures are seemingly ever increasing due to both an increase in the aging population and a rise in obesity rates, contributing to the increase in bone fractures. Permanent implants are expected to serve for the whole term of a patient's life, in contrast to temporary implants, which are required for a shorter time period to allow for the healing of broken bones [1]. Current orthopaedic implants of both categories are produced from metallic materials, which display adequate mechanical properties and fatigue and corrosion resistance; however, the stiffness of metallic materials is greater than that of bone, and this can cause stress shielding [2,3]. As a less stiff alternative to metal implants, aliphatic polyesters are a group of biocompatible and bioresorbable polymers used in a wide range of biomedical applications. Some of the most commonly studied polymers include polylactide (PLA), polycaprolactone (PCL) and polyhydroxyalkanoates (PHA) [4–6].

Each of these polymers has advantages and disadvantages for biomedical applications [7]. Poly l-lactide (PLLA) has been used in a range of medical devices [8], including various orthopaedic clinical applications [9], where its favourable mechanical properties when compared to other biopolymers offers a performance advantage. PLLA has a high tensile strength, low ductility and is a semi-crystalline polymer. The crystalline structure can make PLLA mechanically superior when compared to other polyesters in load-bearing applications [4,9–11]. PLLA has glass transition and melting temperatures of 55–80 ◦C

**Citation:** Naseem, R.; Montalbano, G.; German, M.J.; Ferreira, A.M.; Gentile, P.; Dalgarno, K. Influence of PCL and PHBV on PLLA Thermal and Mechanical Properties in Binary and Ternary Polymer Blends. *Molecules* **2022**, *27*, 7633. https:// doi.org/10.3390/molecules27217633

Academic Editors: Marek Brzezi ´nski and Małgorzata Ba´sko

Received: 23 September 2022 Accepted: 4 November 2022 Published: 7 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

and 170–180 ◦C, respectively. In contrast, polycaprolactone (PCL) is a soft, semi-crystalline polymer with a low melting and glass transition temperatures (55–60 ◦C and −54 ◦C, respectively) [12,13]. Although it possesses high ductility (with a tensile elongation at break of over 700%) and a high impact strength, it has a low tensile strength (~23 MPa) and Young's modulus [3,5,14–16]. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is an aliphatic biodegradable polyester with a melting temperature between 80 and 160 ◦C and a glass transition temperature in the range of −5 to 20 ◦C depending on the HV (hydroxyvalerate) content of the copolymer, which can be adjusted to control the mechanical properties and degradation of the polymer [17]. PHBV has been shown to produce consistent favourable bone tissue adaptation response in addition to the elimination of any undesirable chronic inflammatory responses (up to 12 months after implantation) [18].

Both PLLA and PHBV are hard polymers with poor impact performance [4,5,9,19], which limits their use. The brittle nature of the polymers can be improved through blending with soft ductile polymers [12]. Blending of polymers is a simple yet effective method to obtain new materials with enhanced properties, as the limitations of the dominant component in the blend can be mitigated. Tuning of the physical and mechanical properties of a blend can be achieved with the selection of appropriate materials, adjustment of the blend compositions and appropriate preparation conditions [14]. The blending of PLA/PCL and PLA/PHBV as two co-polymer blends for use in biomedical applications has been previously investigated [6,20–23]. These studies have shown that it is possible to increase the fracture toughness or elongation at break of PLLA by blending with PCL or PHBV, but with reduced modulus or tensile strength [12,24–26]. Ternary blends of the three materials have not been previously assessed; however, the three materials together offer an attractive combination: PLLA providing strength, PCL giving ductility and PHBV enhancing biocompatibility of the material whilst also contributing to the mechanical properties [27].

Ternary blends with polymers which are not established biomaterials have previously been considered in terms of their ability to modify the behaviour of PLLA, PCL and/or PHBV using (i) polypropylene carbonate (PPC) in PLLA/PHBV/PPC blends [28], which showed reduced strength and modulus compared to PLLA alone; (ii) poly(butylene succinate) (PBS) in PLLA/PHBV/PBS blends [29], which showed reduced strength and modulus but increased elongation at break compared to PLLA alone; and (iii) montmorillonite (MMT) nanoclay in PLA/PCL/MMT-nanoclay blends [30], which increased modulus and strength compared to the PLLA/PCL blend, but reduced toughness and impact strength. These studies reinforce the observation from previous studies with PLLA/PCL and PLLA/PHBV blends that a formulation which provides increased ductility without sacrificing modulus and strength has yet to be found.

The aim of this study was, therefore, to characterise binary and ternary blends of PLLA, PCL and PHBV produced through twin screw extrusion for their suitability for use in orthopaedic applications in terms of mechanical performance and processability. Changes in microstructure are assessed via FTIR and DSC, with mechanical properties characterised in tension using modulus, strength, creep rate and viscosity as the key indicators of processability and mechanical behaviour over extended time periods. As orthopaedic devices are commonly applied for extended time periods, the creep behaviour of the blended materials was considered an important property to consider, and one which had not been studied in previous work on blended polyester formulations.

## **2. Materials and Methods**


*2.1. Study Design and Participants*

From October 2020 to August 2021, we conducted this cross-sectional study in the Department of Neurology and Department of Geriatrics at University Hospital No. 1 in Bydgoszcz, Poland. The study included people who met the inclusion criteria: aged 65 years and older, voluntarily agreed to participate, with sufficient speech, hearing, and cognitive abilities: no dementia or Alzheimer's disease diagnosed by a psychologist or physician. The total population of the respondents was 347.

After admission to the ward, each patient underwent psychological and neurological assessment in order to exclude cognitive impairment and dementia. The standard tool used by the psychologists in Poland is the Montreal Cognitive Assessment test and Mini-Mental State Examination and the Clock Drawing Test. All patients who met the inclusion criteria became participants in this study. As scheduled admissions were on hold for a long time during the COVID-19 pandemic, hospitals operated on an ER, and the number of hospitalized patients was very limited. Access to other hospitals was also restricted. Thus, we were able to collect only such a group of respondents.

Due to the fact that the study was conducted during the epidemic, we took special precautions. The subjects were patients of two departments: the Department of Neurology and Department of Geriatrics at University Hospital No. 1 in Bydgoszcz. Consequently, we spoke to each test person alone in a separate room. All test persons prior to admission to the hospital tested negative for SARS-CoV-2. Each of the study participants and the researchers wore masks. During the meeting, a distance of at least 2 meters was kept. In addition, all completed questionnaires were placed in a specially prepared box, where they

had a grace period of about 7 days. Each of the participants completed the questionnaires independently. In the event of any questions or doubts, the researcher was at his disposal.

### *2.2. Variables and Measurements*

Before the start of the project, a pilot study was carried out on a group of 46 people in order to obtain information on the understandability of the questions included in the Authors-Designed Questionnaire (ADQ). All comments, opinions and suggested changes have been considered. Therefore, we have removed or changed some text items to the final, easy-to-understand form. The results of the pilot studies were not included in the results of this work.

The dependent variables include: elder abuse: psychological, physical, sexual and economic abuse and the risk of EA. The definitions of these variables were:

In addition to the VASS scale, the study also used: ADQ, the Geriatric Depression Scale (GDS) 15 items [17,18] and the Activities of Daily Living (ADL) Scale [19,20]. ADQ was created specifically for the purpose of this study, as no gold standard tool for assessing elder abuse has been published in Poland so far. This tool was developed on the basis of the researcher's own experience in conducting this type of research and the available literature [9,10,13,21,22]. The reliability of the ADQ was examined by computing internal consistency coefficients. The Cronbach alpha coefficient was 0.91. Sociodemographic questions were included in the 1st part of the questionnaire and concerned: sex, age, education, marital status, family income, and place of residence. The leading question was "During the COVID-19 pandemic, have you experienced any abuse (e.g. kicking, pulling, hitting, ridiculing, pushing, insulting) in your place of residence?". As for the various forms of EA, the respondents answered the question: "Which of the following forms of elder abuse were used against you?" selecting from the list of the abuses they have experienced. Above, in the definition of each type of violence, we have listed all the acts characteristic of a given sub-type of abuse, which were included in the ADQ. The next questions concerned, among others: the occurrence of chronic diseases, assessment of one's health condition, feeling lonely, depressed or anxious, and having children.


**Appendix A**

**Table A1.** The Interview Guide Used for the Current Study.

#### **Interview Guide**

**Table A2.** Examples of Statements for Each Individual Category within the Main Themes.

**Table A2.** *Cont*. **Main Themes and Categories Example Codes** • Expected future course of mental health *I hope so and I think so, right or not. It all depends. I think when I find a way and have a goal of what I'm going to do next, I feel better. But without the goal and the plan what will become of me then I feel bad. This uncertainty of what's going to happen to you. It will be better for me here in Germany, I think. Things will be a little bit better. Yeah. Even not with money or anything but with my life. Yeah. I think it will be better here* (B) Access to the psychosocial walk-in clinic in PHV • Barriers *Only the difficult thing is, to meet him, because it is from Monday in the morning and Wednesday in the afternoon. And sometimes, if you come in the afternoon late, you could not meet him because many people are there. Yes,* . . . *I was kind of afraid that people would find out and then laugh at me because I'm here with a psychologist now (yes). Yes.* • Facilitating factors *Yes, I, I thought I (..) I will be helped. I will be able to sleep better, feel better. So he got that from his interpreter.  That is a great relief for us.* (C) Perception of counseling sessions • Helpful *Yeah, she encouraged me and listened to me. I have everything is going to be okay They have motivated me. You have made me brave. I also liked the way they* . . . *which medications I need,* . . . *I also liked that. And the exercises that in some situations where I have so quite stress that I calm down a bit. They have applied to the social welfare office and the court that I am allowed to go to my family.* • Difficulties *No, nothing seemed strange to us, completely normal, just like us. He has difficulties just talking about it, so he wants to say it but he realizes (* . . . *) it's hard for him. So he finds it hard to talk about it in general.* . . . *he says since we have cultural differences, it would be much better that he goes to a doctor, for example, that he has Persian background. And he can understand him better. So now with the translator and the language and then the different questions, which for him sometimes also has no sense, because it just does not fit to his culture The noise that she actually hears while she's sort of in this consultation, that sort of messes her up sometimes.* (D) Perception of follow-up treatment • Motivation *I would like to, because I think it is very important and it affects many African people. They don't know this. Yeah. I never been to a psychiatric doctor before in my life. So this is important. And I will be going all the time. If I would continue to have my problems, then I will* . . . *. see a psychologist No. I hope God is the one who gives health.* • Barriers *Only that others find out and then laugh at me.* . . . *Because I've been bullied so often, I'm really so afraid of it (laughs). He just doesn't know because he can neither speak German nor English how he will get to it later.* • Facilitating factors *If we were allowed to stay in Germany or were not deported, we could make use of all (.) help. There are enough human rights in Germany in this regard.*


2.3.1. Network Structure

A modified atrous spatial pyramid pooling (ASPP) module from DeeplabV3 was connected after the backbone in this branch for capturing the multi-scale context. To fit the size of the feature map from the backbone, the original ASPP module with a dilation rate of (6, 12, 18) was modified to a larger module with a dilation rate of (4, 11, 18, 25). Moreover, the number of output channels of each layer was promoted from 256 to 512. The overall structure of the foreground extraction branch is shown in Figure 3.

**Figure 3.** Structure of the foreground extraction branch.


**13. Application to antimicrobial activity**

The cell wall of bacteria not only precludes any interaction between the glycoconjugates on their membrane and carbohydrate-binding proteins but also prevents these proteins from

Antimicrobial Activity of Lectins from Plants 163

from *Shigella flexneri* and *Salmonella abortivoequina*. In 1970, other investigators demonstrated that Concanavalin A can be used to detect lipopolysaccharides of various Salmonella strains

In 2010, Petnual et al. reported the antimicrobial activity of *Curcuma longa* lectin, expressed as the minimal inhibitory concentration (MIC), was found to inhibit the growth of all five microbial species tested, the four bacteria, *Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa*, plus the yeast *Candida albicans*, at MIC values of ≥ 0.011, 0.005, 0.092, 0.002 and 0.0046 mg/ml, respectively (Petnual et al. 2010). These results demonstrate that the *Curcuma longa* rhizome lectin is likely to be at least one of the, if not the, candidate molecule responsible for the antibacterial action observed in rhizome extracts from this plant. An outstanding feature of the antibacterial activity of the isolated lectin is it is somewhat nonselective against this fairly diverse selection of bacteria. The potentially broad effect of the *Curcuma longa* rhizome lectin on the growth inhibition of several diverse bacterial strains, confirms the important interaction between the lectin and all the strains under consideration. From the tested strains, *Pseudomonas aeruginosa* (lowest MIC) seemed to be most sensitive to the presence of lectin. Previouse studies of the binding of plant lectins to bacterial cell wall peptidoglycans indicate that several lectins of different carbohydrate specificities can recognize most of the components of the bacterial cell wall, such as muramic acid, *N*acetylglucosamine, *N*-acetylmuramic acid and muramyl dipeptide (Ajouba et al., 1994).

Archidendron jiringa seed lectin was selected to test for antimicrobial activity with *Escherichia* coli, Pseudomonas auroginosa, Bacillus subtilis, Staphylococcus aurous, and Candida albican (Charungchitrak et al., 2011). The MIC of Archidendron jiringa seed lectin with Candida albican equal in S. aurous to be 0.0567 mg /ml and in Bacillus subtilis to be 0.2266 mg/ ml. But the MIC with *Escherichia* coli and Pseudomonas auroginosa is not detected, demonstrating stronger antimicrobial activity against gram-positive than gramnegative bacteria. Accordingly, the binding of lectins to muramic acid and N-acetylmuramic acid, carbohydrates present in the bacterial cell wall (mainly in gram-positive bacteria), has been reported (Ajouba et al., 1994). These data suggest that lectins probably play a role in plant defense, not only against phytopathogenic invertebrates, herbivores or fungi, but also against bacteria. The carbohydrate-binding site probably plays a key role in this activity, being responsible for the recognition of bacteria. Almost all microorganisms express surfaceexposed carbohydrates. These carbohydrates may be covalently bound, as in glycosylated teichoic acids to peptidoglycan, or non-covalently bound, as in capsular polysaccharides (Hirmo, et al., 1997; and Caldeon, et al., 1997). Every surface-exposed carbohydrate is a potential lectin-reactive site. The ability of lectins to form complexes with microbial glycoconjugates has made it to be employed as probes and sorbents for whole cells,

The lectin from *Curcuma amarissima* inhibited 4 microbial growth consist of *Bacillus subtilis*, *Candida albicans*, *Escherichia coli*, and *Staphylococcus aureus* at concentration ≥ 0.446, 0.446, 0.223, and 0.892 mg/ml respectively. But can not inhibite *Pseudomonas auroginasa* growth because at the surface of *Pseudomonas auroginosa* cell does not have polysaccharide ligands which can interact with *Curcuma amarissima* lectin. (Kheeree et al., 2011) Similar to Legume lectin from *Trinella foenumgraecum, Trifolium alexandrium, Bauhinia variegata,* and *Delonix regia* had a research that these lectins from sephadex G-150 can agglutinated both gram negative and gram positive bacteria (*Mycobacterium rhodochrous, Bacillus cercur, Bacillus megaterium,*

as determined by gel diffusion. (Goldstein and Staub, 1970).

mutants, and numerous cellular constituents and metabolites.

penetrating the cytoplasm. Therefore, plant lectins cannot alter the structure and/or permeability of the membrane or disturb the normal intracellular processes of invading microbes. Therefore, if lectins play a role in the plant's defense against bacteria, it must be through an indirect mechanism that is based on interactions with cell wall carbohydrates or extracellular glycans.

In 1936, a using lectin in clinical microbiology began when Summer and Howell (Summer and Howell, 1936) had a report that Concanavalin A can agglutinated various *Mycobacterium* spp. The interactions between plant lectin and microorganisms have been applied for typing of bacteria, fungi, and protozoa. It is useful for characterizing bacterial cell components and for detecting bacteriophage receptors. (Etzler, 1983; Lis and Sharon, 1986; and Nicolson, 1974). The unique property of lectin to bind non-covalently to simple sugars and polysaccharides has attracted interest in microbial taxonomy. Lectin has a role in the clinical laboratory identification and taxonomic classification of many microorganisms. Because lectins are generally monoclonal proteins and because they possess a spectrum of specificities and molecular weights, they are substantial tools for diagnostic microbiology applications. Recent observations with regard to the binding of plant lectins to components of the bacterial cell wall peptidoglycans (such as muramic acid, *N*-acetylmuramic acid, *N*acetylglucosamine and muramyl dipeptides) revealed that seed lectins from several legume species strongly interact with these bacterial surface carbohydrates (Ajouba et al., 1994). Evidently, the observation that legume seed lectins can recognize and bind to the bacterial cell wall does not imply that such an interaction occurs *in vivo* and certainly does not prove that these lectins are involved in the protection of the seedlings against bacteria.

Lectin has been used for investigating virulence factors, surface structures, and identification of gram-positive bacteria. For example; lectin from *Dolichos biflorus* was used to confirm its specificity for identifying group C streptococci. In another test, its crude extract was coupled to polystyrene particles with a spacer arm to yield an effective lectinlatex reagent that agglutinated group C streptococcal antigens prepared as nitrous acid, autoclave,or enzyme extracts. (Slifkin and Gil, 1984) Group C streptococcal isolates from horses and cattle agglutinated with lectin from *Dolichos biflorus* and *Helix pomatia*. (Schalla et al., 1986). Concanavalin A could be precipitated various bacterial polysaccharides, with interacts specifically with bacterial cell walls containing glycosidic residues associated with teichoic acid. Accordingly, bacteria teichoic acids from cell wall containing α-glucopyranisyl residues,such as *Lactobacillus plantarum*, *Staphylococcus aureus*, and *Bacillus subtilis*. (Archibald and Coapes, 1971; Doyle et al., 1982; and Reeder and Ekstedt, 1971). Lectins from soy bean have been used to assay for detecting *Bacillus anthracis* (Cole et al., 1984). The use of soybean agglutinin (SBA) to detect very low numbers of buffered suspention of *Bacillus anthracis* vegetative cells and spores has been reported (Graham et al., 1984). The strategy was to bind the cells or spores to polystyrene plates and to detect the bound forms with horseradish peroxidase labeled soybean agglutinin (called the lectinosorbent assay).

The contrast of gram-negative bacteria and gram-positive bacteria is the cell wall of gramnegative bacteria contains to lipid but cell wall of gram-positive bacteria does not have the lipid. In 1968, Doyle et al., provided evidence that Concanavalin A reacts with macromolecules that are devoid of terminal glucopyranose or mannopyranose residues (Doyle et al., 1968). Their investigations demonstrated that Concanavalin. A precipitates lipopolysaccharide preparation derived from various strains of *Escherichia coli* as well as

penetrating the cytoplasm. Therefore, plant lectins cannot alter the structure and/or permeability of the membrane or disturb the normal intracellular processes of invading microbes. Therefore, if lectins play a role in the plant's defense against bacteria, it must be through an indirect mechanism that is based on interactions with cell wall carbohydrates or

In 1936, a using lectin in clinical microbiology began when Summer and Howell (Summer and Howell, 1936) had a report that Concanavalin A can agglutinated various *Mycobacterium* spp. The interactions between plant lectin and microorganisms have been applied for typing of bacteria, fungi, and protozoa. It is useful for characterizing bacterial cell components and for detecting bacteriophage receptors. (Etzler, 1983; Lis and Sharon, 1986; and Nicolson, 1974). The unique property of lectin to bind non-covalently to simple sugars and polysaccharides has attracted interest in microbial taxonomy. Lectin has a role in the clinical laboratory identification and taxonomic classification of many microorganisms. Because lectins are generally monoclonal proteins and because they possess a spectrum of specificities and molecular weights, they are substantial tools for diagnostic microbiology applications. Recent observations with regard to the binding of plant lectins to components of the bacterial cell wall peptidoglycans (such as muramic acid, *N*-acetylmuramic acid, *N*acetylglucosamine and muramyl dipeptides) revealed that seed lectins from several legume species strongly interact with these bacterial surface carbohydrates (Ajouba et al., 1994). Evidently, the observation that legume seed lectins can recognize and bind to the bacterial cell wall does not imply that such an interaction occurs *in vivo* and certainly does not prove

that these lectins are involved in the protection of the seedlings against bacteria.

horseradish peroxidase labeled soybean agglutinin (called the lectinosorbent assay).

The contrast of gram-negative bacteria and gram-positive bacteria is the cell wall of gramnegative bacteria contains to lipid but cell wall of gram-positive bacteria does not have the lipid. In 1968, Doyle et al., provided evidence that Concanavalin A reacts with macromolecules that are devoid of terminal glucopyranose or mannopyranose residues (Doyle et al., 1968). Their investigations demonstrated that Concanavalin. A precipitates lipopolysaccharide preparation derived from various strains of *Escherichia coli* as well as

Lectin has been used for investigating virulence factors, surface structures, and identification of gram-positive bacteria. For example; lectin from *Dolichos biflorus* was used to confirm its specificity for identifying group C streptococci. In another test, its crude extract was coupled to polystyrene particles with a spacer arm to yield an effective lectinlatex reagent that agglutinated group C streptococcal antigens prepared as nitrous acid, autoclave,or enzyme extracts. (Slifkin and Gil, 1984) Group C streptococcal isolates from horses and cattle agglutinated with lectin from *Dolichos biflorus* and *Helix pomatia*. (Schalla et al., 1986). Concanavalin A could be precipitated various bacterial polysaccharides, with interacts specifically with bacterial cell walls containing glycosidic residues associated with teichoic acid. Accordingly, bacteria teichoic acids from cell wall containing α-glucopyranisyl residues,such as *Lactobacillus plantarum*, *Staphylococcus aureus*, and *Bacillus subtilis*. (Archibald and Coapes, 1971; Doyle et al., 1982; and Reeder and Ekstedt, 1971). Lectins from soy bean have been used to assay for detecting *Bacillus anthracis* (Cole et al., 1984). The use of soybean agglutinin (SBA) to detect very low numbers of buffered suspention of *Bacillus anthracis* vegetative cells and spores has been reported (Graham et al., 1984). The strategy was to bind the cells or spores to polystyrene plates and to detect the bound forms with

extracellular glycans.

from *Shigella flexneri* and *Salmonella abortivoequina*. In 1970, other investigators demonstrated that Concanavalin A can be used to detect lipopolysaccharides of various Salmonella strains as determined by gel diffusion. (Goldstein and Staub, 1970).

In 2010, Petnual et al. reported the antimicrobial activity of *Curcuma longa* lectin, expressed as the minimal inhibitory concentration (MIC), was found to inhibit the growth of all five microbial species tested, the four bacteria, *Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa*, plus the yeast *Candida albicans*, at MIC values of ≥ 0.011, 0.005, 0.092, 0.002 and 0.0046 mg/ml, respectively (Petnual et al. 2010). These results demonstrate that the *Curcuma longa* rhizome lectin is likely to be at least one of the, if not the, candidate molecule responsible for the antibacterial action observed in rhizome extracts from this plant. An outstanding feature of the antibacterial activity of the isolated lectin is it is somewhat nonselective against this fairly diverse selection of bacteria. The potentially broad effect of the *Curcuma longa* rhizome lectin on the growth inhibition of several diverse bacterial strains, confirms the important interaction between the lectin and all the strains under consideration. From the tested strains, *Pseudomonas aeruginosa* (lowest MIC) seemed to be most sensitive to the presence of lectin. Previouse studies of the binding of plant lectins to bacterial cell wall peptidoglycans indicate that several lectins of different carbohydrate specificities can recognize most of the components of the bacterial cell wall, such as muramic acid, *N*acetylglucosamine, *N*-acetylmuramic acid and muramyl dipeptide (Ajouba et al., 1994).

Archidendron jiringa seed lectin was selected to test for antimicrobial activity with *Escherichia* coli, Pseudomonas auroginosa, Bacillus subtilis, Staphylococcus aurous, and Candida albican (Charungchitrak et al., 2011). The MIC of Archidendron jiringa seed lectin with Candida albican equal in S. aurous to be 0.0567 mg /ml and in Bacillus subtilis to be 0.2266 mg/ ml. But the MIC with *Escherichia* coli and Pseudomonas auroginosa is not detected, demonstrating stronger antimicrobial activity against gram-positive than gramnegative bacteria. Accordingly, the binding of lectins to muramic acid and N-acetylmuramic acid, carbohydrates present in the bacterial cell wall (mainly in gram-positive bacteria), has been reported (Ajouba et al., 1994). These data suggest that lectins probably play a role in plant defense, not only against phytopathogenic invertebrates, herbivores or fungi, but also against bacteria. The carbohydrate-binding site probably plays a key role in this activity, being responsible for the recognition of bacteria. Almost all microorganisms express surfaceexposed carbohydrates. These carbohydrates may be covalently bound, as in glycosylated teichoic acids to peptidoglycan, or non-covalently bound, as in capsular polysaccharides (Hirmo, et al., 1997; and Caldeon, et al., 1997). Every surface-exposed carbohydrate is a potential lectin-reactive site. The ability of lectins to form complexes with microbial glycoconjugates has made it to be employed as probes and sorbents for whole cells, mutants, and numerous cellular constituents and metabolites.

The lectin from *Curcuma amarissima* inhibited 4 microbial growth consist of *Bacillus subtilis*, *Candida albicans*, *Escherichia coli*, and *Staphylococcus aureus* at concentration ≥ 0.446, 0.446, 0.223, and 0.892 mg/ml respectively. But can not inhibite *Pseudomonas auroginasa* growth because at the surface of *Pseudomonas auroginosa* cell does not have polysaccharide ligands which can interact with *Curcuma amarissima* lectin. (Kheeree et al., 2011) Similar to Legume lectin from *Trinella foenumgraecum, Trifolium alexandrium, Bauhinia variegata,* and *Delonix regia* had a research that these lectins from sephadex G-150 can agglutinated both gram negative and gram positive bacteria (*Mycobacterium rhodochrous, Bacillus cercur, Bacillus megaterium,*

Antimicrobial Activity of Lectins from Plants 165

In 2011, Kheeree et al. purified *Curcuma amarissima* lectin showed *in vitro* antifungal activity against three plant pathogenic fungal species, *Colectrotrichum cassiicola*, *Exserohilum turicicum*, and *Fusarium oxysporum*. It strongly inhibited the growth of *Colectrotrichum cassiicola* at 17.5 µg for *Fusarium oxysporum* and *Exserohilum turicicum*, which were strongly inhibited at the higher concentration of 35 μg (Fig. 6). Antifungal activity has been observed in other lectins where, for example *Astragalus mongholicus* root lectin revealed antifungal activity against various species of phytopathogenic fungi (Yan et al., 2005). Similarly with lectin from *Talisia esculenta* seeds inhibited the growth of *Fusarium oxysporum*, *Colectrotrichum lindemuthianum*, and *Saccharomyces cerevisiae.*(Freire et al., 2002) *In vitro* studies demonstrated that two novel chitin-binding lectins seeds of *Artocarpus integrifolia* inhibited the growth of *Fusarium moniliforme* and *Saccharomyces cerevisae* (Trindade et al., 2006). Many studies of plant lectins have assumed that they are implicated in host defense mechanism as antifungal proteins. However, to date only a small number of lectins have been reported to have actual antifungal activity such as lectin from the rhizomes of *Ophiopogon japonicus* showed antifungal activity against *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008). The purified *Phaseolus coccineus* Lectin (PCL) was devoid of antifungal activity against *Candida albicans* and Penicillium *italicum* (Chen et al., 2009).

Fig. 6. Inhibitory effect of purified *Curcuma amarissima* lectin on the *in vitro* growth on PDA plates (as an antifungal activity bioassay) of; (A) *Colectrotrichum cassiicola*, (B) *Fusarium oxysporum* and (C) *Exserohilum turicicum*. For each plate, 0.625 cm diameter discs were seeded with 10 μl of TBS (a) alone as the negative control, or containing either (b) 17.5 μg/

A large body of data exists on the interaction of lectins with a relatively broad spectrum of parasites ranging from the protozoa through the metazoa. Although Concanavalin A was used by many investigators as a lectin probe for these organisms, many other lectins have been shown to be of value in the study of cell surfaces and the identification and differentiation of the parasites. In some instances virulence of parasitic protozoa appears to be related to their surface properties, as revealed by interactions with lectins. Thus, several investigators have deemed important the comparison of surface saccharides of parasites known to differ in their virulence traits. It has been conjectured that the virulence of the trophozoite form of *Entamoeba histolytica* may depend, in part, on its surface properties. Data have been presented indicating that only strains isolated from cases of amoebic dysentery agglutinate with Concanavalin A (Martinez-Palomo et al., 1973) strains isolated from asymptomatic cases of amoebic dysentery, however, do not agglutinate with this lectin.

ml or (c) 35 μg/ ml purified *Curcuma amarissima* lectin (Kheeree et al., 2011).

*Bacillus sphaericus, Escherichia coli, Seratia marcescens, Corynebacterium xerosis,* and *Staphylococcus aureus)* (Reda et al., 1992). Inaddition β-galactoside-binding lectin was extracted from the skin of amphibian, *Bufo arenarum*. It had an antimicrobial activity against Gram negative bacteria (*Escherichia coli* K12 4100 and wild strains of *Escherichia coli* and *Proteus morganii*) and Gram positive bacteria (*Enterococcus faecalis*) (Alicia *et al*., 2003).

Several investigators have concluded that lectins are useful reagents for the study of fungal cell surfaces and may also be of value as important aids in the classification of fungi (Barkai and Sharon, 1978). The major components of fungal cell wall is Chitin, a polymer of β-(1, 4)-*N*-acetyl-D-glucosamine (Barkai and Sharon, 1978; and Ebisu et al., 1977). The report of lecin interaction to fungal, such as fluorescein-conjugated wheat germ agglutinin has been shown to be an effective probe to detect chitin on hypha surfaces.  In 1975 Mirelman et al. was found wheat germ agglutinin (WGA) can be inhibits spore germination and hyphal growth of *Trichoderma viride* and interferes with the synthesis of chitin (Mirelman et al., 1975). A novel mannose-binding lectin was purified from rhizomes of *Ophiopogon japonicus* was showed antifugal activity in three phytopathogenic fungi namely *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008).

In 2010, Petnual et al. purified *Curcuma longa* lectin at a dose of 47 μg and 94 μg/0.3 cm2 disc showed antifungal activity against the three tested phytopathogenic fungal species, *Exserohilum turicicum*, *Fusarium oxysporum* and *Colectrotrichum cassiicola* (Fig. 5). While the lectin dose of 47 μg/0.3 cm2 disc slightly inhibited the growth of these three fungi, that at 94 μg/0.3 cm2 disc showed a higher and significant degree of antifungal activity on all three isolates (Petnual et al. 2010). This effective lectin dose of around 100 μg/ 0.3 cm2 disc is in accord with that reported for the lectin from *Annona muricata* seeds against the growth of *Fusarium oxysporum*, *Fusarium solani* and *Colletotrichum musae* (Damico, et al., 2003), and for the lectin from *Astragalus mongholicus* against *Fusarium oxysporum, Colletorichum* sp. and Drechslera *turcia* (Yan, et al., 2005). Other lectins, such as those from potato (Gomez, et al., 1995) and red kidney beans (Ye, et al. 2001), have also been reported to exhibit antifungal activity. However, novel non-lectin proteins with antifungal activity in plant rhizomes are also known, such as the 32 kDa protein in ginger rhizomes which exhibits antifungal activity toward *Fusarium oxysporum* at a dose of 32-160 μg/ 0.3 cm2 disc of ginger rhizome (Wang and Ng, 2005).

Fig. 5. Inhibitory affect of *Curcuma longa* lectin on antifungal protein toward *Exserohilum turicicum* (A), *Fusarium oxysporum* (B) and *Colectrotrichum cassiicola* (C). The negative control is 10 μl of 20 mM Tris-HCl buffer pH 7.4 (a), 47 μg *Curcuma longa* lectin. (b) and 94 μg *Curcuma longa* lectin (c) (Petnual et al., 2010).

*Bacillus sphaericus, Escherichia coli, Seratia marcescens, Corynebacterium xerosis,* and *Staphylococcus aureus)* (Reda et al., 1992). Inaddition β-galactoside-binding lectin was extracted from the skin of amphibian, *Bufo arenarum*. It had an antimicrobial activity against Gram negative bacteria (*Escherichia coli* K12 4100 and wild strains of *Escherichia coli* and

Several investigators have concluded that lectins are useful reagents for the study of fungal cell surfaces and may also be of value as important aids in the classification of fungi (Barkai and Sharon, 1978). The major components of fungal cell wall is Chitin, a polymer of β-(1, 4)-*N*-acetyl-D-glucosamine (Barkai and Sharon, 1978; and Ebisu et al., 1977). The report of lecin interaction to fungal, such as fluorescein-conjugated wheat germ agglutinin has been shown to be an effective probe to detect chitin on hypha surfaces.  In 1975 Mirelman et al. was found wheat germ agglutinin (WGA) can be inhibits spore germination and hyphal growth of *Trichoderma viride* and interferes with the synthesis of chitin (Mirelman et al., 1975). A novel mannose-binding lectin was purified from rhizomes of *Ophiopogon japonicus* was showed antifugal activity in three phytopathogenic fungi namely *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008).

In 2010, Petnual et al. purified *Curcuma longa* lectin at a dose of 47 μg and 94 μg/0.3 cm2 disc showed antifungal activity against the three tested phytopathogenic fungal species, *Exserohilum turicicum*, *Fusarium oxysporum* and *Colectrotrichum cassiicola* (Fig. 5). While the lectin dose of 47 μg/0.3 cm2 disc slightly inhibited the growth of these three fungi, that at 94 μg/0.3 cm2 disc showed a higher and significant degree of antifungal activity on all three isolates (Petnual et al. 2010). This effective lectin dose of around 100 μg/ 0.3 cm2 disc is in accord with that reported for the lectin from *Annona muricata* seeds against the growth of *Fusarium oxysporum*, *Fusarium solani* and *Colletotrichum musae* (Damico, et al., 2003), and for the lectin from *Astragalus mongholicus* against *Fusarium oxysporum, Colletorichum* sp. and Drechslera *turcia* (Yan, et al., 2005). Other lectins, such as those from potato (Gomez, et al., 1995) and red kidney beans (Ye, et al. 2001), have also been reported to exhibit antifungal activity. However, novel non-lectin proteins with antifungal activity in plant rhizomes are also known, such as the 32 kDa protein in ginger rhizomes which exhibits antifungal activity toward *Fusarium*

*oxysporum* at a dose of 32-160 μg/ 0.3 cm2 disc of ginger rhizome (Wang and Ng, 2005).

Fig. 5. Inhibitory affect of *Curcuma longa* lectin on antifungal protein toward *Exserohilum turicicum* (A), *Fusarium oxysporum* (B) and *Colectrotrichum cassiicola* (C). The negative control is 10 μl of 20 mM Tris-HCl buffer pH 7.4 (a), 47 μg *Curcuma longa* lectin. (b) and 94 μg

*Curcuma longa* lectin (c) (Petnual et al., 2010).

*Proteus morganii*) and Gram positive bacteria (*Enterococcus faecalis*) (Alicia *et al*., 2003).

In 2011, Kheeree et al. purified *Curcuma amarissima* lectin showed *in vitro* antifungal activity against three plant pathogenic fungal species, *Colectrotrichum cassiicola*, *Exserohilum turicicum*, and *Fusarium oxysporum*. It strongly inhibited the growth of *Colectrotrichum cassiicola* at 17.5 µg for *Fusarium oxysporum* and *Exserohilum turicicum*, which were strongly inhibited at the higher concentration of 35 μg (Fig. 6). Antifungal activity has been observed in other lectins where, for example *Astragalus mongholicus* root lectin revealed antifungal activity against various species of phytopathogenic fungi (Yan et al., 2005). Similarly with lectin from *Talisia esculenta* seeds inhibited the growth of *Fusarium oxysporum*, *Colectrotrichum lindemuthianum*, and *Saccharomyces cerevisiae.*(Freire et al., 2002) *In vitro* studies demonstrated that two novel chitin-binding lectins seeds of *Artocarpus integrifolia* inhibited the growth of *Fusarium moniliforme* and *Saccharomyces cerevisae* (Trindade et al., 2006). Many studies of plant lectins have assumed that they are implicated in host defense mechanism as antifungal proteins. However, to date only a small number of lectins have been reported to have actual antifungal activity such as lectin from the rhizomes of *Ophiopogon japonicus* showed antifungal activity against *Gibberella saubinetii* and *Rhizoctonia solani* (Tian et al., 2008). The purified *Phaseolus coccineus* Lectin (PCL) was devoid of antifungal activity against *Candida albicans* and Penicillium *italicum* (Chen et al., 2009).

Fig. 6. Inhibitory effect of purified *Curcuma amarissima* lectin on the *in vitro* growth on PDA plates (as an antifungal activity bioassay) of; (A) *Colectrotrichum cassiicola*, (B) *Fusarium oxysporum* and (C) *Exserohilum turicicum*. For each plate, 0.625 cm diameter discs were seeded with 10 μl of TBS (a) alone as the negative control, or containing either (b) 17.5 μg/ ml or (c) 35 μg/ ml purified *Curcuma amarissima* lectin (Kheeree et al., 2011).

A large body of data exists on the interaction of lectins with a relatively broad spectrum of parasites ranging from the protozoa through the metazoa. Although Concanavalin A was used by many investigators as a lectin probe for these organisms, many other lectins have been shown to be of value in the study of cell surfaces and the identification and differentiation of the parasites. In some instances virulence of parasitic protozoa appears to be related to their surface properties, as revealed by interactions with lectins. Thus, several investigators have deemed important the comparison of surface saccharides of parasites known to differ in their virulence traits. It has been conjectured that the virulence of the trophozoite form of *Entamoeba histolytica* may depend, in part, on its surface properties. Data have been presented indicating that only strains isolated from cases of amoebic dysentery agglutinate with Concanavalin A (Martinez-Palomo et al., 1973) strains isolated from asymptomatic cases of amoebic dysentery, however, do not agglutinate with this lectin.

Antimicrobial Activity of Lectins from Plants 167

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The unique property of lectins to bind noncovalently to simple sugars and therefore to polysaccharides and glycoconjugates has attracted the interest of virologists. In virology, lectins have been used for detection of viral glycoproteins in purified and infected cells, as well as for viral purification. Lectin studies have revealed information about the structure of viral glycoproteins, structures important in their pathogenicity. A significant contribution of lectin use in virology has been in the development of unique diagnostic methods that yield specific identification of viral agents. Purified influenza virus yields macroscopically visible flocculation when mixed with Concanavalin A. (Klenk et al., 1984) When influenza virus is treated with a proteolytic enzyme, the glycoprotein spikes of the virus are released. These treated viral particles no longer agglutinate with this lectin, but will flocculate in the presence of *N*-acetylgalactosamine-associated lectins, such as *Dolichos biflorus* or *Helix pomatia*. Other viruses, including arboviruses, vesicular stomatitis virus, paramyxoviruses, leukoviruses, and hepatitis B virus, also agglutinate with Concanavalin A. Concanavalin A was shown to block specifically adsorption of the bacteriophage binding sites of *Bacillus subtilis* possessing αglucosylated teichoic acids in the cell walls associated with teichoic acids. It was suggested that the application of this lectin might be useful as a means to correlated bacteriophage and serologic typing of staphylococci. (Archibald and Coapes, 1972).