Molecular Dynamics Simulation and Analysis of some Ligands on Var2csA Target - Juniper Publishers

 Novel Approaches in Drug Designing & Development - Juniper Publishers  

Abstract

Prevalence of malaria during pregnancy and the spate of drug resistance by malaria parasites have constantly impacted maternal, perinatal and neonatal outcomes, especially in sub-Saharan Africa. Inhibiting binding; or displacement of bound infected erythrocytes from the placenta as an adjunct treatment or vaccine for malaria was considered an option towards ending pregnancy associated malaria in this study. Molecular modeling and toxicity predictors used in this study indicated that among the ligands screened, IH3 had the lowest binding energy of -9.8Kcal/mol while var2csA had -2.8Kcal/mol. Var2csA is parasite’s adhesive protein. It was also observed that out of the 90 ligands (binding affinity range -9.8 to -1.0 Kcal/mol) screened, IH3 (-9.8Kcal/mol), FAD (-8.4 Kcal/mol), NDP (-8.2 Kcal/mol), A5A (-8.2 Kcal/mol), ABO (-8.1 Kcal/mol), IH2 (-7.8 Kcal/mol), 2RT (-7.7 Kcal/mol), CRO (-7.7 Kcal/mol) and IH1 (-7.7 Kcal/mol) appear to be the most promising lead compounds to occupy var2csA binding pocket in pCSA in order to prevent adhesion of malaria infected erythrocytes to the placenta. SwissADME and Molinspiration Cheminformatics for LogP (mean of 1.07 and range of -2.79 to 4.18) of the lead compounds showed no correlations between lipophilicity and interaction with receptors. Of all the compounds selected for analysis, only ABO and 2RT exhibited drug-like properties based on Ghose, Lipinski and Veber filters. The data therefore suggests that IH3, FAD, NDP, A5A, ABO, IH2, 2RT, CRO, IH1 and var2csA make favourable lead candidates for targeting pCSA and therefore require further in vitro and in vivo evaluations.

Keywords: Malaria; Pregnancy; Modeling; Toxicity; Auto Dock

Abbreviations: pRBCs: Parasitized Red Blood Cells; IEs: Infected Erythrocytes; DC: Domain Cassette; EPCR: Endothelial Protein C Receptors, PAM: Pregnancy Associated Malaria; DARC: Duffy Antigen Receptor for Chemokines; EC: Endothelial Cell; IPT: Intermittent Preventive Treatment; PDB: Protein Data Bank; TEST: Toxicity Estimation Software Tool; FAD: Flavin Adenine Dinucleotide

Introduction

Background to the Study

Malaria is one of the leading causes of death in tropical, underdeveloped countries throughout the world. Malaria in pregnancy is a major global public health concern; this has been attributed to transient depression in cell-mediated immunity which has increased the susceptibility of pregnant women to malaria and its adverse outcomes. Approximately 32.3% of pregnant women in sub-Saharan Africa are at the risk of malaria yearly [1]. Malaria is caused by Plasmodium sp transmitted by the bite of the female Anopheline mosquito. Upon infection, the life cycle of the Plasmodium sp. begins and ends in the bloodstream where erythrocytes are infected and consequently destroyed, causing symptoms such as anemia, respiratory sequelae, cerebral malaria, metabolic acidosis, and eventually organ failure, leading to death [2]. However, before the total destruction of erythrocytes occurs, there seems to be a mode by which the parasitized red blood cells (pRBCs) become sequestered or adhered in particular sites throughout the vascular system [3] and this results in widespread sequestration of infected erythrocytes (IEs) and hence their reduced clearance from the blood stream by the spleen [4]. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates this adhesion of IEs to various host receptors on the vascular lining during the blood stage of malaria infection [4-6].

Proteins of the PfEMP1which include var2csA (DBL2x, DBL3x and DBL6ɛ), Var1csa (DBL3γ), CIDR groups B & C, CIDR of DC8 group A, CIDR of DC13, DC4, DC8, DC5, Duffy Binding Protein (DBP) for Pv & Pk, and PfRh4 for Pf, PfRh5, EBA - 175, EBL -1, EBA - 140 and EBA 181 mediate adhesion and cell invasion through specific binding to multiple endothelial cell (EC) receptors, including domain cassette (DC) 36, DC31also known as platelet endothelial cell adhesion molecule 1 (PECAM -1) or cluster of differentiation 31 (CD31) [7], DC13, DC8, intercellular adhesion molecule -1 (ICAM - 1), E-selectin, endothelial protein C receptors (EPCR), duffy antigen receptor for chemokines (DARC), and placental chondroitin sulfate A (pCSA). Others include: CD36, complement receptor - 1 also known as CD35 (i.e.; C3b, or C4b), VLA-4, LW, CD 58, CD 147, CD44, B-CAM/LU and Glycophorin A (F actin and G actin).

In this study, we focused on pregnancy associated malaria (PAM) also referred to as placental malaria (PM) which is mediated by Plasmodium falciparum elaborated var2csA that binds to placental chondroitin sulfate A (pCSA) to bring about sequestration of malaria IEs on the placental cyncytiotrophoblast. Each Plasmodium falciparum parasite genome constitutes over 59% var genes [5,8] and individual parasite expresses a single var gene at a time [9], maintaining all other var genes in a transcriptionally silent state. Almost all members of the var family are classified into one of 3 major groups (A, B, and C) based on a combination of chromosomal location, transcription direction, and upstream promoter sequence [10]. As an important activity in many drug discoveries projects, in silico molecular modeling was employed to identify molecules with activity against pCSA and subsequent lead optimization of the hits identified [11].

Statement of the Problem

Use of insecticide treated net and intermittent preventive treatment (IPT) are currently the preventive strategies to improve maternal and fetal outcomes amidst the ravaging malaria scourge. However, due to waning efficacy of the IPTs and insecticide treated nets as a result of resistant strains of the parasite and the malaria transmitting vectors, these preventive measures have become ineffective and less reliable over-time. This problem has persisted for a while now and there is no valid vaccine yet to prevent malaria, especially, placental malaria [12].

In this study, our target is to identify ‘‘in silico”, for the purpose of clinical application, non-recombinant product(s) that can competitively inhibit Plasmodium falciparum infected erythrocytes (PfIEs) form binding, or displace bound PfIEs from placental chondroitin sulfate A since no antibody or antigen based vaccine tend to be successful in the search for vaccine against pregnancy associated malaria.

Aim and Objectives of the Study

Aim

This study aims to discover “in silico” non recombinant molecules which can interact with placental chondroitin sulfate to inhibit binding or displace bound var2csA from the placenta in order to prevent pregnancy associated malaria.

Objectives

1. To generate a library of molecules similar to var2csA (ligands to CSA) from available databases.

2. Dock these molecules against placental CSA in order to generate hits with favorable binding affinity with it; and

3. Analyze “in silico” the suitability and safety of some of the selected molecules as drugs.

Operational Definition of Terms

1. Autodock: Molecular modeling simulation software. It is especially effective for protein–ligand docking.

2. Binding affinity: The strength of the binding interaction between a single biomolecule to its ligands.

3. Databases: A collection of information that is organized so that it can be easily accessed, managed, and updated. Example here include PDB and PubChem.

4. Virtual molecular screening (also known as molecular docking): A computational technique used in drug discovery to identify those structures which are most likely to bind a drug target, typically a protein or enzyme.

Literature Review

Concept of Drug Design and Receptor Interaction

Many micro and macroscopic properties of substance, such as biological activities of drugs and drug-like compounds are controlled by their stereochemistry. Molecules with one or more rotational degree of freedom (rotatable bonds) exhibit conformational isomerism, where the stable conformers correspond to energy minima, and therefore, to the most probable structures to be found in materials, pharmaceuticals and agrochemicals [13]. Interaction stabilizing conformations are provided by certain hydrogen bonding even though others also take place in many cases, as seen in electrostatic effects and hyperconjugation phenomena, i.e, electron donation from the endocyclic oxygen lone pair toward the vicinal axial antibonding orbital [14]. For example, the core of the CSA-binding site is situated in the N-terminal part of var2csA by hydrogen bonding and van der Waal force (Figure 1). The following equation [EI ]aq [E]aq +[I ]aq is used to determine the reaction involving a receptor and its ligand. The change in free energy of binding (ΔG) is related to binding affinity, ( ΔG = −RTInk_A).

Where E = Receptor, I = Ligand, K_A = Equilibrium constant, R = ideal gas constant = 8.314J/mol-K, and T = Temperature of the reaction (Kelvin).

Receptor and ligand docking aim at correct prediction of the structure of the complex under equilibrium conditions. The docking techniques used in this study included prediction of the binding modes of novel drugs (ligands) and the study of proteinprotein interactions using AutoDock Vina (http://vina.scripps. edu) tool in PyRx (http://pyrx.sourceforge.net) where autodock reproduces nine different protein - ligand complexes for which the structure of the macromolecule was known without prior knowledge of the binding site. The more negative the binding affinity becomes, (i.e, the lower the binding energy), the better the orientation of the ligand in the binding pocket of the receptor.

The autodock programme package - PyRx was able to select the correct complexes based on the energy without prior knowledge of the binding site; (i.e, blind docking). PyRx was not able to see the binding site but was able to find it.

Prediction of correct structure (posing) of the [EI ] complex does not require information about kA. Prediction of biological activity (ranking) does require information about K_A . For the complex [EI ] , the following factors are important: steric, electrostatic, hydrogen bonding inhibitor strain (if flexible) and enzyme (receptor) strain. Important factors when considering the equilibrium include: desolvation, rotational entropy, and translational entropy. Most scoring functions focus on energetic than entropic effects. The events of ligand-binding are driven by a combination of enthalpic and entropic effects, either of which can dominate specific interactions [15-17].

Var2csA

Var2csA is a variant of many adhesive proteins of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) elaborated on the surface of Plasmodium falciparum infected erythrocytes for the purpose of tethering infected cells on tissue receptors to evade destruction by the spleen. Var2csA protein consists of six DBL domains, a single transmembrane helix, cytopasmic acidic terminal sequence and significant interdomain regions (Figure 2). Four of the DBL domains have been shown to bind CSA [18-20].

The structure of the DBL3X domain has been shown [22,23] to have an ɑ-helical scaffold similar to those observed in DBL domains from the invasion proteins like erythrocyte-binding antigen 175 (EBA-175) and the Duffy antigen receptor for chemokines (DARC) - binding protein [24,25].

It has also been shown that full length recombinant var2csA binds specifically to CSA with nanomolar affinity, and that the CSA- binding site lies in the N-terminal part of the protein [26]. The core CSA- binding site lies within the DBL2X domain and part of the flanking interdomain regions, in contrast to the idea that single domains do not possess the structural requirement for specific CSA binding. They added that the x-ray scattering measurement revealed CSA- binding DBL2X domain is situated in the center of the structure.

Materials and Methods

Materials

The following computing resources and items were used:

Compounds: The protein data bank (PDB) structure of the receptor chondroitin sulfate A (PDB ID: 3bqk) in its raw and prepared forms is shown in Figure 4. Structures of some of the ligands used are shown in Figures 3

Hardware and Software: This work was carried out on windows 8.1 Pro with processor: Intel® Core (TM) i5 CPU M 520 @ 2.40GHz having installed memory (RAM): 4.00GB (3.86 GB usable) on system type: 64 bit operating system although 32-bit Windows Vista operating system also work well. PyRx docking software version 0.8 for Windows [14]. PyRx is open source software to perform virtual screening. It is a combination of several softwares such as AutoDock Vina, AutoDock 4.2, Mayavi, Open Babel, etc.

PyRx uses Vina and AutoDock 4.2 as docking softwares. In this study, AutoDock Vina [27] was used. Discovery studio (Discovery Studio: v20.1.0.19295), Pymol (Pymol stereo 3D quad buffer) and ICM Browser (Molsoft MolBrowser 3.8-7d) were used to examine structural properties and study binding interaction between receptor residues and ligands. SwisADMET, and Molinspiration cheminformatics were used for evaluation of drug-likeness of the ligands and Toxtree [28] was additionally used to determine the toxicity and safety profile of the ligands.

Databases and Applications: Protein data bank (rcsb.org) and PubChem (https://pubchem.ncbi.nlm.nih.gov/) were used to download the chemical structures of the receptor (3bqk) and those of the ligands. Canonical SMILES and other information about the ligands and the receptor were extracted from PubChem. Toxtree, Toxicity Estimation Software Tool (TEST), SwissADMET, Molinspiration and Lazar Toxicity Predicter were employed to test various toxicity and safety parameters of lead compounds.

Methods

Retrieval of Macromolecule: The structure of the macromolecule-chondroitin sulfate A (CSA) was retrieved by searching in the protein data bank (PDB) (rcsb.org/ structure/3bqk); downloaded and saved as a PDB format. Retrieval of Liands: Molecules constituting the ligands screened belong to the same enzyme commission (EC) class of CSA and were downloaded from rcsb.org as Excel file. In the file, the ID values of all the class members were copied and pasted on a page “download ligand” site in the PDB in order to generate their corresponding ideal structure data file (SDF) formats. The “launch download” application of PDB downloaded and channeled the ligands into a folder (ligands) that was created on the desktop. Some of the ligands are presented in Table 1.

Target Preparation and Docking Process: The x-ray structure of the receptor (3bqk) was downloaded from protein data bank (rcsb.org), cleaned from hetatoms in discovery studio and resaved (Figure 4). The input ligand files were also prepared for virtual screening by converting them to PDBQT file format when they were imported into PyRx software as chemical table file (SDF). Following a series of steps, the library of the ligands was docked into the active site of 3bqk in the PyRx platform using Amber Van der Waals in the active box of 17 x 15 x 17. Autodock Vina took each ligand and bonded its different conformations to the receptor molecule (3bqk) to get the binding energies in different orientations of each ligand. Each ligand has nine different binding orientations starting from 0 to 8.

Validation of Docking Process: Docking was repeated three times on the same system specifications for the purpose of process validation and all returned minimal variation (P<0.01 data not shown) in uff energy, binding energy and RMSD values.

Binding Analysis: Discovery studio (Discovery Studio: v20.1.0.19295), Pymol (Pymol stereo 3D quad buffer) and ICM Browser (Molsoft MolBrowser 3.8-7d) were all used to analyze binding interactions between residues of 3bqk and the ligands.

Drug likeness: Toxtree, Toxicity Estimation Software Tool (TEST), SwissADMET, Molinspiration and lazar toxicity predictions were variously employed to test toxicity and safety parameters of lead compounds.

Results and Discussion

Results

Analysis of Docking Result: A library of 90 compounds in the same EC class with chondroitin sulfate A, known to interact with pCSA was generated from rcsb.org. Compounds in the library demonstrated good binding affinity with many having higher binding affinity to pCSA (3bqk) than 2y8d a var2csA molecule and SO4, a functional species in chondroitin sulfate A (Table 2).

The first 75 ligands and SO4 have binding affinity higher than that of 2Y8D. This implies that var2csA can be displaced from pCSA in preference for molecules which have higher binding affinity. 2Y8D and SO4 which have their highest binding affinity as-2.8Kcal/mol and -3.7Kcal/mol also ranked 719th and 655th respectively. For this study, only the first nine ligands with the highest binding affinity (-9.8 to-7.7Kcal/mol), 2y8d and SO4 were selected for the target analysis. The complex formed for some of the ligands are represented in figure 5, the complexes have different structural modification from the SDF format seen in Figure 4 except for SO4 which it is not a valid selection for binding analysis. It is not a chain, molecule or a multiple residue object.

Analysis of Binding Interactions: Receptor residues interacting with SO4, 2y8d and the top nine selected ligands as seen in Discovery studio and Pymol are listed in Table 3a.

Evaluation of Drug likeness and Organ Toxicity of some Selected Hits: The lipophilicity for selected compounds is as shown by the values of their consensus logP in Table 4. Drug likeness of the compound’s Tables 5a & 5b.

Discussion

All the ligands in this study yielded their lowest binding energy at their first binding modes (0). The reliability of this study was ascertained by triple - docking of the ligands with the receptor (pCSA) and it was found that both the first, second and the third dockings maintained the same values in all parameters and for all binding modes (P < 0.01 data not shown), giving IH3 and FAD as the first two ligands with the lowest binding energy as shown in table 2 and implicitly, the ligands of choice to bind placental chondroitin sulfate during in vitro and in vivo evaluations. The binding affinities of the top nine compounds on var2csA target are closely related that they lie within a close range of -9.8 to -7.7Kcal/ mol indicating that their potential as lead compounds for 3bqk is comparable. Auto Dock reproduces nine different protein -

ligand complexes for which the structure of the macromolecule was known, without prior knowledge of the binding site but here only the result from the first binding orientation was presented since it is the one with the highest binding affinity, the full binding orientations and their RMSD values for all the ligands are available on request. The RMSD for the first binding orientation is always 0. The binding affinity and the RMSD value collectively determine the ranking of the molecules.

Analysis of Binding Pattern

Considering their binding interactions, FAD had the highest number of favorable hydrogen bonds with receptor residues but shares equal number of hydrophobic contacts with IH3 (Table 3a & Table 3b). FAD is a drug already in used to treat eye diseases caused by vitamin B2 deficiency, such as keratitis and blepharitis (Drugbank, http://www.drugbank.ca/). Some other compounds like NDP, ABO, IH2, CRO, IH1 and SO4 possess no hydrogen bonds but have good number of other hydrophobic contacts except CRO and SO4 which do not have any significant favourable bonds with receptor residues based on observation from Discovery Studio (Figures 6).

Drugability of Compounds

No correlation was observed between lipophilicity and the interaction with the receptor by considering cLogP values and number of conventional bonds (Tables 3b & Table 4). However, for FAD, NDP and A5A, interaction with the receptor is correlated with low lipophilicity while 2RT has high cLogP value. The compounds with high cLogP values used their polar functional groups to interact with the receptor. FAD, 2Y8D and IH3 exhibited good hydrogen bond interaction with var2csA receptor of placental chondroitin sulfate A possibly due to their polarity.

Screening the compounds for drug likeness using Lipinski, Veber and Ghose filters, FAD, A5A and IH1 did not satisfy the requirements for a safe drug in all the filters used. However, only ABO and 2RT suitably satisfied the conditions for drug likeness in all the filters and therefore likely the safest (Table 5b). Notwithstanding these observations, all the compounds selected can be relatively safe since the unsatisfied molecules tend to violate 1 or 2 conditions in 1 or 2 of the three filters. Again, FAD which violated almost all the conditions in all the three filters is already in the market for other medical uses.

Based on toxicity prediction of some compounds in Lazar Toxicity Predicter, FAD, NDP and ABO penetrate the human blood brain barrier (Table 6) indicating possibility of central nervous system toxicity. But this could also be an advantage for the compound to access the brain to elicit their pharmacologic activity in the event of susceptible cerebral malaria. They also tend to be non-carcinogenic in mouse model based on observations from the same platform.

Summary of Findings, Conclusion, and Recommendations

Summary of Findings

In this study, an extensive search was carried out in silico to discover non recombinant substances that could serve as vaccines or adjunct treatment for pregnancy associated malaria (PAM) also known as placental malaria (PM). Ligands (or small molecules or drugs) with activity against chondroitin sulfate were extracted from designated drugs and proteins databases. The ligands were screened against a receptor (pCSA, PDB ID: 3BQK) to isolate molecules that can favourably bind it thus displacing or inhibiting binding of var2csA, a Plasmodium falciparum (P.f) protein usually deposited on the surface of P.f- infected erythrocytes that enhances the adhesion of the parasite carrying cells to the endothelium of tissue vasculature, thereby tethering them away from splenic destruction.

Ninety ligands were screened out of which 75 were found to have higher binding affinity with pCSA than var2csA, with binding affinity ranging from -9.8 to -2.8 (Table 2, complete data not shown). Out of this number, the best nine ligands (IH3, FAD, NDP, A5A, ABO, IH2, 2RT, CRO and IH1,) whose binding affinities ranged from -9.8 to -7.7 were isolated for further in silico evaluations. Their analysis showed that four compounds (IH3, FAD, A5A and 2RT) had hydrogen (strong covalent) bond interaction with 3bqk while five (NDP, ABO, IH2, CRO and IH1) had no hydrogen bond with 3bqk. One compound (CRO) does not have both hydrogen and hydrophobic bonds with 3bqk, yet it has high binding affinity with the receptor. This was possible because, the compound may have used its polar functional groups to make the interaction. The same is applicable to SO4 (a sulfate ion) which also do not have any hydrogen or hydrophobic contact with the receptor. 2Y8D had both hydrogen bond and hydrophobic bond interactions with the receptor (Table 3a & Table 3b).

In silico drug ability testing of the 9 selected compounds including SO4 and 2y8d indicates that only four compounds (ABO, 2RT, CRO, and 2Y8D) based on Lipinsky, Veber and Ghose rules perfectly satisfied drug likeness conditions (Table 5b). That means 2Y8D can be administered to mimic the parasitic form of it in binding placental chondroitin sulfate A. FAD violated all the rules for a safe drug but it has been approved for use in the treatment of eye diseases caused by vitamin B2 deficiency, such as keratitis and blepharitis. Lazar Toxicity Predicter (Table 6) showed FAD, NDP and ABO to have ability to cross blood brain barrier and non-carcinogenic.

Conclusion

Following the analysis and observations so far, all the 9 selected compounds including SO4, 2y8d and all the rest of the compounds screened deserve further in vitro and in vivo evaluations to determine which compound has the best in vivo activity against placental malaria and safe for human use.

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Kahneman’s Ideas Applied to Dementia: Understanding and Managing Behavior - Juniper Publishers

 Gerontology & Geriatric Medicine - Juniper Publishers

Introduction

As clinicians it is not only important to know which clinical interventions work, but also why they work. When we understand the building blocks and processes underpinning an intervention, we can improve it based on the theory. This article examines the behaviors of people with dementia using Kahneman’s (2011) notion of ‘fast and slow’ thinking. Kahneman’s ideas help explain why people with dementia can continue to perform complex activities into late stage dementia (knitting, ironing, playing a musical instrument, riding a bike, driving a car). The ‘fast and slow’ model can guide us on how to support skilled activities and to interrupt processes that might lead to illbeing (e.g. repetitive questions or acts).

Understanding the processes underpinning a typical behavior in dementia

Maria, a woman with dementia, has the intention to go to the shops to buy a crusty loaf. She leaves her home with this firm intention, but an hour later we find her knocking at the door of a house where she had lived 10 years earlier. She is shouting and demanding to see her child. This type of memory failure is common in people with dementia. It is important to try to understand the mechanisms underlying memory failure because they provide information about how to deal with distress and maintain wellbeing in people with dementia.

If we had observed her during this trip, we would have noticed Maria making a number of choices as she went on her journey. Her first choice point came when she arrived at the junction of her street, she should have turned left to go to the shops but instead turned right. Having turned right, she eventually came to the entrance to the park. She opted to go into the park. After a short walk she exited the gates on the far side, and then she decided to take the no. 5 bus. This took her near her son’s ‘old’ school. She alighted from the bus but found the school closed and so progressed to the house where she had previously lived.

Ignoring the fact that Maria has dementia for a moment, her behavior reveals something important about the way humans process information. This is most eloquently described in the work of Kahneman [1] in his notion of ‘fast and slow’ processing. Kahneman suggests that slow thinking is conscious deliberate thought, whereas fast thinking is quick and often unconscious processing. The two features operate together whereby the slowed thinking creates the conscious goals, and ‘checks in’ every so often to ensure our plans are on track. In contrast, the fast-automatic processing enables us to carry out routine activities without using too much effort or thinking power. In other words, it allows us to ‘run in autopilot’ – see box 1.

Box 1

In terms of Maria, we see her engaging in fast and slow thinking, but her poor short-term memory means she has an inability to remember and focus on her original goal (getting the crusty loaf). Therefore, instead of basing her decisions on this goal, they are based on cues that she is encountering on her journey (e.g. the no.5 bus, school, vicinity of house). She is ‘running on auto-pilot’, relying on system 1 to do a lot of fast, automatic decision-making based on her environment. During this time, her slow system 2 keeps ‘checking-in’ to see if she is ‘on track’ but cannot remember what the goal is. She is moving around like a pin-ball in a pin-ball machine, making new goals and decisions based on whatever information is available to her. A representation of Maria’s information processing is shown in (Figure 1).

The final part of Maria’s journey is particularly important because here we see evidence of her remembering a goal over a sustained period of time (i.e. the desire to see her son). This goal was cued by passing his old school. In her time-shifted state, she thinks her son is a schoolboy and wants to collect him. However, because the building is closed, she panics and decides to go home (‘old’ home) to find him. This is relevant because she seems able to retain this goal for a longer period of time than she was able to remember that she wanted to get a crusty loaf. This is almost certainly because of the high level of emotion attached to seeing her son, as emotional memories are retained more easily for people with dementia.

Clinical Relevance

The above scenario, and its theoretical underpinnings, are relevant to clinical practice. Indeed, in addition to having explanatory power in relation to Maria’s journey, it offers guidance in two other important areas (i) the de-escalation of challenging behaviors (CB), and (ii) the selection of treatments in dementia.

(i) De-escalation of CBs – In a typical de-escalation situation people with dementia have got a particular goal and their attempt to fulfil this may put themselves, or someone else, at risk. However, the caregiver’s attempt to intervene may be perceived by someone with dementia as interfering or over-controlling which can further inflame the situation and lead to aggression.

Such a situation is about to happen in Maria’s scenario because she is now trying to find her son, and bangs on a stranger’s door; the goal and drive to see her son is strong. The poor unsuspecting occupant of the house, Aidan, is about to encounter a difficult situation. If he responds to Maria in a way that further upsets her, the situation could become problematic and he may get assaulted. Fortunately, however, Aidan works at the local care home and is skilled in dealing with such situations. He recognizes Maria has a dementia and guesses correctly that she is time-shifted. Hence, he knows that a direct confrontation of her current view would be unhelpful. He is aware that he needs to ‘go along’ with her current view of reality for a while and look for an opportunity to grab her attention with a powerful topic of distraction. By doing this he knows that he will have a chance to re-orientate her to the present and the current reality.

Aidan invites Maria into the house and says she can wait to see if her son comes home. Despite being agitated she sits and accepts a cup of tea. Aidan’s friendly chat calms her and reduces her ‘emotional thinking and her drive’ somewhat. He notices that she is taking a lot of interest in his Labrador and asks does she like dogs. Maria is a big dog lover and she is soon talking enthusiastically about the many dogs she’s owned. Aidan is hugely attentive, asking lots of questions about her dogs. Five minutes later Maria has been distracted from her son. Aiden doesn’t want Maria to be reminded of her son again, so he asks his own children to stay out of the room until her taxi arrives.

Aidan did something really simple here, something that many caregivers do without recognizing the processes underpinning it. Aidan’s choice of topic, and the enthusiastic manner he used to engage Maria, resulted in a shift from ‘children’ to ‘dogs’. We can see how fast and slow thinking underpins this interaction because we can understand how the CB stems from cues in the environment altering her goals (slow thinking) and short-term decisions (fast thinking). In this situation, Aiden is able to use this to his advantage by encouraging Maria to focus on different, more helpful cues like his dogs. This redirected her slow thinking to forget about the goal of seeing her son so that her fast processes were anchored in the current situation rather than focused on this outdated goal. He also removed cues which could retrigger her distressing thoughts of her son.

In clinical practice we can use this idea to:

a) Identify what elements of the environment are triggering the goals and thoughts behind CBs

b) Remove these cues to prevent CBs.

c) Introduce topics and cues that can redirect attention to more situation-appropriate thoughts and goals which are anchored in the present (or away from the distressing thoughts).

(ii) Selection of treatments in dementia – The term treatment can be confusing because it could refer to interventions to either reduce the rate of the dementing process or to tackle agitation or aggression. However, in the current discussion the interventions are designed to maintain wellbeing.

Unfortunately, the evidence base for effective wellbeing treatments is rather inconsistent, except in case of Cognitive Stimulation Therapy (CST) and some person-centered programs [2]. In part, this is due to the fact that dementia includes a wide range of diseases which may be at many different stages, and also that the experience of dementia is different for every person. This means that studies which might show positive effects of interventions in some cases are watered down by the fact they may not be appropriate for every individual at every stage.

To deal with such variability, occupational therapist (OTs) have produced a number of programs that attempt to match interventions to the stage of the condition [3]. We suggest that the effectiveness of the OT frameworks can be further enhanced by factoring-in Kahneman’s notion of ‘fast and slow’ processing. Let’s look at the implication of Kahneman’s ideas with respect to people with moderate to advanced dementia.

a) People with dementia have difficulties sustaining activities that require the retention of long-term goals or strategies (slow thinking). Therefore, the use of prompts at key points would be helpful.

b) Activities that include overlearned activities (procedural memories and fast thinking) can be exploited and are able to provide a sense of accomplishment. However, to maintain the person’s active participation he/she may require assistance at transition points between actions which require slow thinking (e.g. when baking a cake the person may struggle with regulating the oven; selection of ingredients; timings).

c) If the tasks fulfil some basic emotional needs (fun, touch, being active, sense of belonging, feeling safe) they are more likely to be engaged initially and their interest and understanding of the activity is more likely to be sustained [4].

Kahneman’s ideas lead us to promote activities that provide positive experiences ‘in the moment’ that do not require goals or strategies (e.g. listening to music, speaking with an attentive listener, looking at photographs, engaging with table-top interactive touch games). It is also helpful to choose actions that provide immediate feedback because this facilitates the use of ‘autopilot’ activities. It is worth noting that the above recommendations are not new and many of them are being done intuitively by experienced cares. However, what is different is that the theory informs us why the approaches are effective. Further, if we start to understand the building blocks of such interventions, we can produce more effective treatments. Therefore, what we are offering here is the theory underpinning the choice of intervention.

An example of this idea can be seen in a study we undertook in 2014 in which we spoke to Christian clerics about their experiences of providing religious services to people with dementia [5]. The most exciting part of this project was to see people with advanced dementia, who appeared to struggle to speak in a residential setting, suddenly become engaged and lively during the religious service. One person attending a Catholic mass fully participated in the prayers, hymns and symbolic aspects of the service. She smiled throughout, seeming to have a sense of belonging in a community from which she had previously appeared detached. Such projects need to be replicated across other religious groups and across other such communal topics. However, the key point being made is that the woman attending the service was able to participate in the service through a mixture of fast and slow processes. She knew the prayers and hymns once they had begun (fast thinking) and was being guided through the transitions by both the structural elements provided by the mass and the behaviors of the other members of the congregation so she was not reliant on slow thinking and retaining information about the service.

‘It is’ and ‘it isn’t’ child’s play – activity in the moment

Before concluding the article, it is worth reflecting on the skills required to devise activities for people with dementia. A good activity for someone with an advanced dementia is something that does not require a goal or strategy – this is where dominoes is better than draughts, which is superior to chess. Further, the actions associated with the task need to be basic and intuitive, another reason dominoes is good (involving a simple matching task), and why the card game ‘snap’ would be more appropriate than either ‘whist’ or ‘poker’.

Also, it is better to use activities that can be undertaken and enjoyed ‘in the moment’, and ones that rely on reflexes: playing simple instruments, ‘copy-cat’ yoga, simple ball games, etc. It is worth noting that games that are traditionally used with small children contain features that make them ideal in late stage dementia. Childhood brain development and brain deterioration have overlapping phases and therefore the types of activities that are suitable for children may be relevant for people with dementia.

It is important to remember however, that unlike children, people with dementia have an array of skills that have been developed in the past that can be utilized if the correct cues are employed. This is well illustrated in the church service example. In this case, a vast range of skills were unlocked once we had provided the person with an environment that supported and triggered the expression of their skills.

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Triage System and Emergency Pediatric Medical Care - Juniper Publishers

 Global Journal of Nanomedicine - Juniper Publishers   

Abstract

Triage system is a process that is critical to the effective management of modern emergency departments. The triage systems aim not only to provide a fair clinical evaluation for the sick, injured and poisoned children, but also to provide a good effective approach to the organization, monitoring and evaluation of emergency medical care in the pediatric emergency departments Over the past 20 years in the world and in Europe, triage systems have been standardized in a number of countries and are making efforts to ensure sustainability of compulsory implementation. In addition, differences at international levels in triad systems, but limits capacities to benchmark standards.

The purpose of this paper is to enable pediatric emergency pediatric healthcare professionals to quickly evaluate, monitor, treat, and transport medical care, and to understand, learn, and apply the newest guidelines of the triage system with priorities. The research of the paper aims to make pediatric emergent medical care professionals in hospital settings to do a fairly fast triage, reducing morbidity, invalidity and mortality in the pediatric emergency department at the hospital. Triage systems in our country at three levels of health care do not work, is a chaotic system because there are no clinical guidelines, algorithms important component should create mandatory triads in three health care providers, advancing, strengthening the triad system as an important component of health care. Education, training of professionals of health care professionals for the three levels of health care should be an obligatory component to increase the quality of emergency medical care in life-threatening children. It’s time to develop and test a triad scheme that will be based on a triad system that will respond and adapt to the conditions of our health system.

Keywords: Triage; Pediatrician; Seriously Ill Critical Children; Convulsive Attacks; Shock; Health Care; Clinical Signs; Life Threatening

Introduction

Pediatric emergency triage system and emergency medical care is a process that is critical to the effective management of modern emergency departments [1]. Trial Systems aim not only to provide a fair clinical assessment for sick, injured and poisoned children, but also to provide a good effective approach to the organization, monitoring and evaluation of emergency medical care in pediatric emergency departments. Over the last 20 years in the world and in Europe, triage systems have been standardized in a number of countries and are making efforts to ensure the sustainability of enforcement [2]. In addition, differences at international level in triage systems but limit the capacity to benchmark. The country-level triage system in chaotic, disorganized Kosovo does not function as a synchronized system but there is no official standard, normative tri-level triage system for health system organization [3].

WHO Emergency triage, assessment and treatment guidelines are used to identify children with life-threatening problems who present with signs and symptoms that should be treated as medical emergencies. But the Kosovo Ministry of Health should bear in mind that this important component should create mandatory triage standards in the three health care needs. It also needs advanced, empowered EMEA and the triage system as an important component of healthcare with education, education, training of health care professionals to enhance the quality of emergency medical care for children at risk.

Purpose of Work

The purpose of this paper is to enable and enable pediatric emergency health care professionals to do rapid triage, assessment, monitoring, treatment, and transport with medical care. To understand, learn, apply the newest triage system guidelines with priority by reducing morbidity, disability and mortality in the hospital pediatric emergency department.

Material and Methods

The research material was obtained from the Children’s Clinic archive at the UCCK Pediatric Emergency Department for the period January - December 2016. The research is retrospective, descriptive, and qualitative. 17 years of age with acute lifethreatening illnesses by researching the pattern of triage, gender, age, place of residence, seasons of the year, monitoring, observation, diagnosis, treatment, complications, referral and education of pediatric emergency medical professionals [4-8]. The sample surveyed 220 cases of children with triad of lifethreatening life-threatening illnesses by pediatric emergency medical professionals with serious illnesses such as febrile convulsive attacks, severe dehydration, hypoxemia, pneumonia, shock, metabolic problem, respiratory distress, cardiac arrest, electrolyte imbalance. Description of data processing Description of data processing is performed statistical parameters, (worked out Exel Word) structure index, arithmetic mean and standard deviation. Statistical tests: X2-test and T-test. Test verification was done for the 95% and 99% confidence levels, respectively for p <0.01 and p <0.05 [9-11].

Results

The research material was obtained from the Children’s Clinic archive at the UCCK Pediatric Emergency Department for the period January - December 2016. The research is retrospective, descriptive, qualitative. Only 28-day-old babies up to the age of 17 with acute life-threatening illnesses treated in Department of Pediatric Emergency of UCCK in Prishtina, out of the total number were 23,320 cases 99.69%, and of them were 327 emergency cases or 1.31%. (Table 1). The average age of the patients included in the study was 1-10 years; the youngest patient was over 28 days and the oldest was a child 16 years (Graph 1) (Table 2) . From the total number of 327 children affected by child sex, male cases were 177 cases or 54.12% and female children 150 cases or 45.88%. (X2- test = 39.4, P <0.001), (Graph 2). The average age of the patients included in the study was 1-10 years; the youngest patient was over 28 days and the oldest was a child 16 years. (Graph 2). Number of cases according to systemic acute life-threatening diseases Neurological 56 cases or 17.12%, Cardiac cases 34 or 10.39%, Respiratory cases 80 or 24.46%, Gastroenterology 125 cases or 38.2%, Metabolic 20 cases or 6.11%, and Toxicological cases 12 or 3.66 % (Table 3).

Number of cases with acute illnesses, children with cardiac arrest 7 cases or 2.23%, asthmatic attack 60 cases or 16.43%, respiratory distress 14 cases 4.22%, convulsive febrile attack 80 cases or 25.43%, shock 18 cases or 5.50%, Diarrhea 118 cases or 36.08%, Hypoglycemia 17 cases or 5.19%, Hyperglycemia 13 cases 4.92% (Graph 3). Number of cases that required emergency medical services with life-threatening illnesses by month of the year [12-16]. January-December with obstructive bronchitis were 49 cases or 15.04%, June-September with diarrhea were 126 cases or febrile conditions were 152 cases or 46.44%. (Graph 4) (Table 4). According to the place of residence, the largest number of cases of potentially life-threatening children were from the city 199 cases or 60.86% and from the village were 128 cases or 39.14% Table 7 (Graph 5). Of the 327 cases reported, only 18 or 5.50% of them were in shock, whereas hypovolemic cases were 8 cases or 44.49%, cardiogenic shock 3 cases or 16.6%, vasodilator shock 2 cases or 11.11%, allergic reactions anaphylactic 1 case or 5.54% and with septic shock 2 cases or 11.19% (Graph 8).

Triage and management of pediatric cases with life-threatening and circulatory injury signs determined based on three clinical criteria such as cold hands and extremities or capillary refills> 3 s and with poor and rapid pulse in all children with these clinical signs have been categorized in children with shock or circulatory impairment [17-21] (Table 5). The number of shock cases defined by three clinical criteria of 18 cases of children with any of the clinical signs of children with cold extremes were 4 cases or 22.23%, capillary duration> 3 s 6 cases or 33.33 and with poor and rapid pulse 8 cases or 44.44% (Table 9) (Graph 9). Children with multiple clinical manifestations of shock may be evaluated in settings restricted by non-specialist healthcare professionals, only with the presence of three clinical signs, and the presence of one or two of these signs indicates a nonspecific impairment of blood circulation that may be due to conditions different from circulatory disorders [22,23] (Graph 6). The total number of 327 cases of them with Respiratory or Obstructive Problems, Absence, Central Cyanosis and Severe Respiratory Disturbances were 74 cases or 22.62%, of them with Respiratory Impairment 61 cases or 82.45%, Respiratory missing 3 cases or 4.05%, central cyanosis respiration 6 cases or 8.10% and respiratory distress respiratory distress 4 cases or 5.40% (Table 9 Graph 9) (Graph 7).

(Graph 7) The total number of 327 cases was 79 cases or 24.15%, with problems of consciousness disorders, whereas 59 cases or 74.68% with consciousness problems and 20 cases or 25.32% with no consciousness problems (Table 6). The total number of 327 cases out of them 118 cases or 36.08%, children with signs of severe dehydration, defined as the presence of diarrhea or any other dehydration condition were with vomiting or insufficient fluid intake 63 cases or 53.40% , with fever were 41 cases or 34.74% and with lethargic or unconscious signs, eyes in orbit, cold skin less capillary filling (> 2 s) were 14 cases or 11.86% (Table 6) (Graph 8). Out of a total of 327 children screened, monitored, observed, diagnosed and treated only needed additional laboratory and diagnostic examinations, biochemical and hematological analysis (hemogram electrolytes, glycemia, urea, creatinine), were 240 cases or 73.39%, Function. lumbal 30 cases or 9.19%, ECG 37 cases or 11.31 and EEG 20 cases or 6.11%.

Blood tests for glucose and sodium recommend that routine blood glucose measurement is not required for all children with febrile convulsions and as the information for the diagnosis from blood cultures of children with febrile convulsions does not differ much from that obtained in the pediatric emergency departments. Of the total number of 327 cases in children is the needed CT scan 109 cases or 33.33%, Magnetic resonance 38 cases or 11.63%, and Radiologic Assist 180 cases or 55.04%. X-ray imaging, computed tomography (CT) or magnetic resonance imaging (MRI), but although CT and MRI devices are often not available in secondary care settings, X-ray skull management is useful in the diagnosis of febrile attack (Table 7) (Graph 5). Use of antipyretic and anticonvulsant drugs in the management of high temperature and convulsive ataxia. The use of antipyretics and anticonvulsants with Ibuprofen were 11 cases or 13.75%, Paracetamol 7 cases or 8.75%, Diazepam rectal shin 37 cases or 46.25, Phenobarbitone Clobazam 25 cases or 31.25%, and drugs respectively. others such as Lorazepam, Valproate, and Phenytoin which are not used at all due to the absence of these drugs (Table 8). The administration of infusions and medications during CPR is considered an important component of critical emergency care of infants and children with serious illness, and fluid use within the first 1-2 hours after initial evaluation.

Isotonic solutions were treated 195 cases or 60.63%, Isotonic solutions containing salt and sugar (NaCl 0.9% and Glucosum 5%) treated 70 cases or 21.8%, hypotonic solutions 0.45% NaCL 0.45% with 5% glucose were treated 12 cases or 4.03%, Adrenaline glucose treated 12 cases or 4.03%, Adenosine treated 7 cases or 2.14%, Amiodarone treated 9 cases or 2.75%, Diuretic treated 12 cases 4.03%, and Verapamil 10 cases or 3.05% were treated (Table 9). Of the total number of 327 cases required was masked oxygen administration in 34 cases or 10.39%, 146 cases monitored or 45.64%, unchecked 20 cases or 6.11%, masked oxygen delivery 36 cases or 11.%, intubated 7 cases or 2.14%, RKP 7 cases or 2.14%, respiratory oxygen delivery 7 cases or 2.14%.

Of the total number of 327 cases with emergency medical care, advanced life care treatment were 575 cases or 17.43%, and treatment with basic medical life care was 270 cases or 82.57%. From the total number of 327 cases with complications were 123 cases or 37.69%, without complications 207 cases or 63.31%. Number of cases after admission, monitoring, observation, medical diagnostics, medical consultations, out of 327 cases hospitalized were 305 cases or 93.27%, after emergency treatment were left home for further treatment. 12 cases or 93.27%. Immediate treatment begins when rapid triage is made and life-threatening medical problems are identified such as: airway, respiratory and circulatory evaluation, ABC in case of injuries, and circulatory diseases, airways and respiratory tract CAB).

Discussion

Over the last 20 years in the world and in Europe, triage systems have been standardized in a number of countries and are making efforts to ensure the sustainability of enforcement. But important, however, are the three most common emergencies such as: respiratory distress (hypoxemia), severe circulation (shock), and consciously disordered convulsive attacks and rapid onset by utilizing triage priorities and a more acceptable model for the country. No uniform triage system exists in the United States (USA), this creates a widespread dilemma of assessing the sick and injured, and each region has its own US emergency medical service network. The lack of a uniform system in the US is in contrast to other industrialized countries such as Canada and Australia. Formation of methods that integrate field and hospital needs will support an emergency medical service system that ensures the best continuity of care.

Nursing (IT) nurses need to adapt the approach and assessment based on the child’s chronological and emotional age, especially when feelings and emotions can predominate and capable of expressing themselves. Barriers exist during nursing triage such as age, very young, older persons generally present more difficulties for inexperienced nurses. Non-hospital and hospital systems - In pre-hospital and hospital settings emergency nursing triage aims to determine the priorities of treatment and transportation of the sick and injured. Design principles and protocols must be developed and promulgated by the appropriate medical authorities. In case of major disasters: earthquakes, floods, fires, severe traffic accidents, mines, where a large number of people can be injured within a short period of time, it is difficult to properly trace because working conditions are similar to those of war

Assessing infants and children is a major challenge but these should be assessed as life signs as in an adult, with except the pulse will drop. in the brachial artery and not in the radial artery. Assessment of vital signs monitoring and recording, detailed examination of the body. A child who is upset, crying or screaming or examining the chest and abdomen will be difficult. But using games or entertaining can help the nurse’s nurse to assess breathing rhythm. The trauma nurse in children should never forget the vital signs, all of which can be recorded on the scene and the triage rooms in the priority cards. From this triage, patients can be sent to treatment rooms according to the severity of the disease. Limited space in the treatment area can cause the triage nurse a lot of problems with labeling priorities which may be incorrectly applied to the waiting room, so a quick retrofit should be done

Rapid triage and rapid, aggressive fluid resuscitation is the basis or cornerstone in hypovolemic and septic shock management; concluding that fluid boluses were potentially harmful to children with signs of severe circulatory damage including shock shock International guidelines and training courses such as advanced life support for children (Mackway-Jones et al., 2005. 2010. 2015) and advanced pediatric life support (Fuchs et al., 2007) provide criteria for clinical signs that if present indicate that a child is in ‘shock’. Febrile seizures are the most common childhood attacks, occurring on average in 4% of children. Three-tiered healthcare systems in our country do not work is a chaotic system because there is a lack of clinical guidelines, algorithms and protocols to make a correct pediatric triage system. It is high time to develop and test a triage scheme or model that will rely on a responsive triage system adapting to the conditions of our health system.

Conclusion

Health care institutions should develop and test an adaptable triage scheme that will rely on a triage system that will respond to and adapt to the conditions of our health system. Department of Pediatric Emergency of UCCK in Prishtina, out of the total number were 23,320 cases 99.69%, and of them were 327 emergency cases or 1.31%. The median age of the patients included in the study was 1-10 years; the youngest patient was over 28 days and the oldest was a child 16 years. From the total number of 327 children affected by child sex, male cases were 177 cases or 54.12% and female children 150 cases or 45.88%. (X2-test = 39.4, P <0.001. Number of cases with acute illnesses, children with cardiac arrest 7 cases or 2.23%, asthmatic attack 60 cases or 16.43%, respiratory distress 14 cases 4.22%, convulsive febrile attack80 cases or 25.43%, shock 18 cases or 5.50%, Diarrhea 118 cases or 36.08%, Hypoglycemia 17 cases or 5.19%, Hyperglycemia 13 cases 4.92%. The number of shock cases defined by three clinical criteria of 18 cases of children with any of the clinical signs of children with cold extremes were 4 cases or 22.23%, capillary duration> 3 s 6 cases or 33.33 and with poor and rapid pulse 8 cases or 44.44%.

The pediatric emergency department triage rooms should be operational and supported with medical equipment, drugs, medical supplies and assistive devices based on international standards of triage. The Ministry of Health of Kosovo should bear in mind that this important component should create a model of training for the needs of our country, standard clinical guidelines, algorithms and protocols to make a correct pediatric triage compulsory triage system correctly. health care, by advancing, strengthening the triage system as an important health care component Education, education, training of health care professionals for the three levels of health care should be an obligatory component to enhance the quality of emergency medical care for children at risk. It is high time to develop and test a conceptual triage scheme or model that will rely on a triage system that responds to the conditions of our health system.

Recommendation

Institutionally support the advancement and empowerment of the triage system as an important component, the necessary pediatric emergency triage rooms. Develop clinical guidelines, algorithms and triage protocols at the three levels of health care. All health care professionals must be educated, trained in ongoing coaching, communication courses, Paediatric basic life support- AED, Pediatric Advanced Life Support, International Trauma Life Support.

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Eggplant Calyx the Newest Alternative Cure for Leishmaniasis Disease - Juniper Publishers

 Journal of Public Health - Juniper Publishers 

Abstract

For centuries, extracts from plants have been used as folk remedies against various health problems [1]. One of the important plants and herbal medicines is eggplant plant, which has been studied extensively, but its cones wasn’t studied thoroughly until now. This study is concerned with the detection and calibration of phenols in the eggplant cones. All the necessary reagents for detection of tannins, flavonoids and chlorogenic acid were positive, and then calibrated phenols with various solvent showed that the ethanolic aqueous extract has the highest percentage of phenol, followed by the aqueous then methanolic extracts. The properties of this extract in terms of its effects on the inhibition of Leishmania parasites were studied. The ethanolic aqueous extract showed a high measure in the rate of inhibition and is equivalent to IC50=39 μg/ml in vitro, the extract was formulated with cream and applied to randomly treated leishmaniasis patients in a double-blind study. The cases were studied in three groups without injection - with injection of glucantime – with liquid nitrogen treatment. Most of the cases have shown improvement or acceleration of improvement since the first week of treatment, confirming the effectiveness and importance of this plant section, which is usually wasted, that is eggplant cones as a new and undiscovered drug with a prospect to treat leishmaniasis.

Keywords: Eggplant cones; Leishmaniasis; Ethanolic aqueous extract; Methanolic extract , Total phenolic content

Introduction

Leishmaniasis is a tropical and subtropical disease caused by an intracellular parasite transmitted to humans by the bite of a sand fly, mainly Phlebotomus and Lutzomyia (Europe, Northern Africa, the Middle East, Asia, and part of South America); exceptionally, transmission has also been reported as a laboratory accident[2]. According to the World Health Organization (WHO), leishmaniasis is one of the seven most important tropical diseases and it represents a serious world health problem that presents a broad spectrum of clinical manifestations with a potentially fatal outcome [3,4]. It is found in all continents except Oceania [3,5] and is endemic in circumscribed geographic areas in North eastern Africa, Southern Europe, the Middle East, South eastern Mexico, and Central and South America.

Leishmaniasis, which is the third in vector-borne parasitic diseases, after malaria and trypanosomiasis, it is of great importance in the world due to the treatment and control difficulties [6]. According to World Health Organization (WHO) data, approximately 20 million people in 98 countries around the world are infected and 350 million people are at risk [7].The parasite is found in amastigote form in the blood and tissues of the vertebrate hosts, and in the promastigote form in the vector sand fly which are invertebrate hosts. Amastigotes are oval or round shaped, 2-4 μm in size and immobile in macrophage cells of vertebrate hosts. When the parasite multiplies in the cell it destroys the cell, and it can be seen individually or in clusters outside the cell [8]. Promastigote form can be detected in the digestive system of the vector. They are single whip shaped or shuttle shaped with a length of 10-20 μm, a width of 1.5-2.5 μm. Promastigotes proliferate in axenic cultures and in the digestive tract of the invertebrate vector [9].

Cutaneous leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania and the genus Phlebotomus. There are two important stages in the life cycle of Leishmania: amastigotes found in humans and other non-human reservoir mammals, and promastigotes found in sand fly. In the vertebrate host the clinical outcome is depending on the parasite strain and the host immune response. The lesions are confined to the skin and to the mucous membrane. A granulomatous response occurs and a necrotic ulcer forms at the bite site. Macrophages containing amastigotes, which may be killed by sensitized lymphocytes were detected in microscopic smear. The lesion may become chronic, usually accompanied by secondary bacterial infection [10]. It has been treated by a chemical and herbal ways and those treatment give some side effects.

This study highlighted a new plant extract from eggplant calyx that has proven effective against leishmaniosis in vitro by achieving IC50 =39 μg/ml and the detection of some active substances in this wasted part of this plant, Eggplant (Solanum melongena L), which is a vegetable crop, economically important, consumed widely in Syria. It is native in South East Asian region and was domesticated over 4000 years ago [11]. Many studies have proved the effectiveness of this fruit in the treatment of many diseases such as diabetes, gonorrhoea, cholera, bronchitis, dyspnea, dysentery, debilitation and the treatment of hemorrhoids [12,13]. thus, this extract were either completely effective treatment or effective adjuvant treatment.

Material and Methods

Plant materials

Eggplants (Solanum melongena) were purchased from local markets in Damascus. The fruit calyx were obtained, dried, and stored away from moisture. Three types of extracts (aqueous ethanol, aqueous, and methanol) were prepared.

Preparation of the extracts

a) Methanolic extract [14]: The methanolic extract was prepared with a Soxhlet extractor. 30 g of the plant sample was extracted by 250 ml of 99% methanol for four hours, after which the extract was collected and dried using a rotary evaporator.

b) Ethanolic- Aqueous Abstract [14]: The ethanolic- Aqueous extract was prepared by a Soxhlet extractor. 30g of the plant sample was extracted with 300 ml of (ethanol- Aqueous) for four hours, after which the extracts were collected and dried using a rotary evaporator.

c) Aqueous extract [15]: 30 g of cone powder is placed in the extraction flask with 200 ml of distilled water and heated under an ascending cooler for 1 hour and the transcript is filtered and evaporated using a rotary evaporator until dryness.

Determination of total phenols (TP):

The Folin-Siocaltolin Tp method uses phenols in tungsten phosphorus molybdate acid in an alkaline medium resulting in a blue solution measured at absorption at a wavelength of 760 nm where a series of the reactions occur by electron transfer of two phenols leading to the formation of blue complexes. [16] Prepare the calibration chain: 0.5 g of gallic acid is dissolved in 10 ml ethanol → supplemented with water up to 100 ml in a calibration balloon, preferably kept in the refrigerator for up to two weeks.

To prepare the calibration curve

Add 0/1/2/3/4/5 and 10 ml of the above phenol solution to a 100 ml calibration balloon and complete the volume with distilled water. Then extend the volume with water. We will have the following concentrations of phenol: 0/50/100/150/250/500 mg / L Gallic acid. We put 20 μl in separate covets and add 1.58 ml distilled water + 100 μL foline reagent and mix well then leave 8 minutes and 30 seconds, then add 300 μL of sodium carbonate and mix well and leave the pair for two hours at 20 degrees Celsius. Absorption is measured of wavelength 760 nm versus bulk (distilled water instead of sample) [17].

Preparation of sodium carbonate solution

20% sodium carbonate solution 200 g of anhydrous sodium carbonate is dissolved in 800 ml of distilled water, boiled then cooled, and after 24 hours drain and complete volume to 1 liter [16].

Anti-promastigote assay using MTT (in vitro assay)

The experiment was designed using a cell culture plate comprising 96 wells where 500,000 parasites were taken from the homeostasis stage in each well within a final volume of 100 RPMI culture medium supported by 10% of the FBS gene. The samples were immunized in a cooled incubator at 26°C. After 48 hours, the parasite viability was tested using MTT approved viability test by treating all wells with a final concentration of 10% of the pigment for 3 hours. At the end of the incubation time, formazan crystals formed with a special solvent (MTT Solvent) The samples absorption was 540 nm wavelength using a microplate reader. The average percentage of viability of each sample is calculated using the following equation:

The half maximal inhibitory concentration of the parasite cell growth IC50 is calculated using the Excel program and plotted the viability curve in terms of the absorbance of all samples by the curve equation we calculate the value of IC50 given the viability values as averages ±standard deviation [18].

Clinical study of the effect of eggplant cones extracts on leishmaniasis:

This study was a randomized double-blind, included 30 patients. the treatment was applied as three cases (without injection - with glucantime injection - with liquid nitrogen) to evaluate synergistic and non-synergistic therapeutic effects. The patients were randomly assigned from the dermatology hospital in Damascus and were provided with the necessary materials from the Faculty of Pharmacy, Damascus University.

The cream containing the herbal extracts was distributed randomly and everyone was asked to use it twice a day throughout the 4-week treatment period. The patients were asked to attend at the hospital for local injection of the glucantime in the lesion, and that was only for severe cases requiring injection. Patients were diagnosed by dermatologists at the Dermatology Hospital in Damascus university by testing the swab of the lesion to confirm laboratory infection by detecting the presence of the parasite. The area of infection was determined by centimetre (1-2-3-4) cm and assessed its change during the treatment period. Both the degree of redness and edema, as well as the severity of sclerosis and the severity of secondary ulcers, if any, were determined using the Likert encoding scale. The clinical observation was weekly.

Results

By comparing our study with Tiwari et al. [19] study tannins were consistent with our study of iron chlorine with the difference that the reaction with lead acetate was positive in the our present study, and in the interactions of flavonoids Tiwari et al. [19] (Table 1). It was limited to reagent Shinoda only, in this present study we had five reactions that all were positive with Chlorogenic acid.

Phenolic titration results

Similar characters indicate that the statistical differences are not significant while the different letters indicate that (Table 2) the statistical differences are significant, and the value of P <0.05 was used to indicate the statistically significant difference. Statistical differences between the values of these extracts is statistically significant. The aqueous ethanolic extract shows the highest phenolic content followed by the aqueous extract then the methanolic extract (Figure 1) (Table 3). By comparing between current study and previous studies we find that total phenolic content of the aqueous extract is (1025 mg /100g) and this value within the specified range which was studied in 33 species of eggplant of different origins and its value was ranges from (740 to 1430 mg gallic acid 100/g) José et al. [20], but this study was on fruit and not on cones. In another study of total phenolic content in the cones of eggplant Diab et al. [19,21-24] it showed a significant difference in value which was greater (2869 mg / 100 g), and the probability of difference is due to the extraction method as the method of preparation was soaking with stirring for two days. The phenolic content of the ethanolic extract in our study was (1180 ± 12.83 mg / 100 g), and in the study Diab et al. [21] the result was (826 mg /100g), meaning that the ratio was higher in the extract of this present study, with the difference in the method of preparation (Table 4 & 5).

The results of detecting the effect on leishmania parasiteThe results of detecting the effect on leishmania parasite

Discussion and Clinical Conclusions

In this present study, the eggplant cones contain phenolic substances, useful in the treatment of many diseases, where the results of the calibration of phenolic substances showed that the ethanolic-aqueous extract has the highest phenolic content followed by the aqueous extract. Each of these components has its own therapeutic effect as Flavonoids so it play a good role in treating damaged and inflamed tissues [25] strengthening the walls of capillary veins, anti-inflammatory, anti-fungal, antimicrobial, antibacterial [26,27], antiviral, anti-cancer, antivascular, anti-allergic, anti-oxidant [28] preventing injuries caused by free radicals by Inhibiting the free radical enzyme, activation and regeneration of antioxidant systems and has role in reducing the permeability and fragility of the veins [29] And The rutin witch founds in the extract is one of the most important types of flavonoids because it reduces the capillary permeability of blood vessels [30]. Tannins have the strongest effect among phenols for their role in strengthening soft tissues, reducing excess secretions [31-33]and repairing damaged tissues [34].

It works at the same time to stop bleeding due to its astringent effect in addition to its antiseptic effect [35] and it has an important role in the protection of inflamed surfaces of human body [36]. It is also considered anti-tumour because it inhibits the proliferation of cancer cells and prevents DNA from vandalism [31,37] .In addition to these properties, tannins have great antioxidant capabilities due to their Phenol nuclei. Most of the antioxidant effects of phenolic compounds are due to its own Oxidation Reduction properties, which make them Reduction agents. Flavonoids and phenolic acids are among the most potent antioxidant compounds. Phenolic acids also have interesting biological properties such as Anti-inflammatory and antipyretic [38], One of these phenolic compounds is chlorogenic acid it has a role as antioxidant [31,33], protect the inflamed surfaces of the mucous membranes, an anti-tumour that inhibits the proliferation of cancer cells and protects the DNA from destruction [39]. With these auxiliary effects of phenols in skin restoration and strengthening of blood vessels, they support the inhibitory effect of leishmaniasis parasites, especially in the ethanolic-aqueous extract. where the correlation in the results was corresponds in terms of the highest rate of phenols and the highest inhibition of leishmaniasis parasites compared with other extracts (Figure 3 -4).

The efficacy in inhibiting the growth of leishmaniasis parasites in the synthetic medium of ethanol-aqueous extract is very close to that of glucantime, the drug traditionally used for the treatment of leishmaniasis. IC50 for the extract =39μg/ml-IC50 for glucantime=35μg/ml [40]. This is consistent with the clinical results of the cream use containing the extract as the healing of cases that require liquid nitrogen and glucantime injection was faster by using the cream and in the case of using it alone the amount of edema gradually reduced and the severity of infection and sepsis significantly lessened from the first week and pictures show that.

Conclusion

We can say that ethanolic- aqueous extract of eggplant cones(calyx), which is neglected in medical and nutritional use showed a high inhibitory effect of Leishmania parasites close in its efficiency to the glucatiem effect. In addition to the presence of active substances in it, including high percentage of phenols, as it helps to repair the affected area and Accelerates its recovery. therefore, the use an effective cream to treat leishmaniasis disease will benefit the patient all over the world which is more economical, easier to apply, without side effect and less painful in the treatment, so this is the goal that we seek to achieve. 

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