In Silico Analysis of the Phenylephrine from the Sand Crab  (Emerita asiatica) for its Antimicrobial Activities

 

Thant Zin1, J. Sivakumar2, C. Shanmuga Sundaram3, U. S. Mahadeva Rao1*

1Faculty of Medicine, Universiti Sultan Zainal Abidin, Terengganu, Malaysia

2PG and Research Department of Biotechnology, Hindustan College of Arts and Science, Padur,

Chennai – 603103

3PG and Research Department of Microbiology, Hindustan College of Arts and Science, Padur,

Chennai – 603103

*Corresponding Author E-mail: raousm@gmail.com

 

ABSTRACT:

Aim: The present investigation gives a detailed report on the analysis of the bioactive compound isolated from the sand crab Emerita asiatica is having better relation with vasoconstriction. Methods: The sand crab Emerita asiatica were collected from Thiruvidandhai beach located on the Kanchipuram District, Tamil Nadu, India, 41 K.M away from Chennai, on the East Coast Road route to Mahabalipuram. Sand crabs were collected by hand picking method. From the muscle mass of the Crab the organic compound was isolated by GC-MS analysis. Molecular docking was also performed to identify the protein ligand responsible for the constriction of blood vessels. Results: The isolated bioactive compound was identified as phenylephrine, an α1 adrenergic receptor which has the potential to lead the molecule for the vasoconstriction. In vitro studies of antibacterial activities clearly indicated that the different concentrations of phenylephrine have the antibacterial activity against the bacteria such as Klebsiella pneumoniae and Staphylococcus aureus. Summary: The present investigation would give latest information on the isolation of bioactive compound for the production of pharmaceutical drugs from marine organisms. These results will be decisive factor for determining a lead bioactive compound for further drug discovery process for the vasoconstriction.

 

KEYWORDS: Emerita asiatica; Klebsiella pneumoniae; Phenylephrine; Staphylococcus aureus; vasoconstriction.

 

 


INTRODUCTION:

There are many species of crabs distributed all over the world. A peculiar species that often misses our eye is the mole crab. This is also called as “sand flea”. Because of their swift digging and burying nature, they are compared to moles. Unlike fleas, they do not cause any bites to humans. Like other crabs they have five pair of legs (Decapods) with the absence of pincers. A characteristic feature of their movement is moving backwards. Females measure 1 inch and are bigger than males (half an inch). Except while feeding, these crabs bury themselves for the entire period of their life 1.

 

The sand crabs, E. asiatica belonging to the family Hippidae are abundant in the inter-tidal sandy beaches of Olcotkuppam (Besant Nagar), Thiruvanmiyur Kuppam, Kanathur Kuppam, Mamallapuram Kuppam and Kalpakkam in Tamilnadu, India. E. asiatica is called as “Elli Poochi” or NaiEluppa Poochi” in Tamil. Prior to tsunami (December 26th 2004) the fisher women used to collect them by hand picking during the low tide period after locating the animals by inserting their feet in the loose sand of the inter-tidal region and sell at a very low price, since the sand crabs are very small containing very little flesh. Usually, the whole animal is ground along with the additives used for preparation of ‘chutney’ a south Indian dish, as in the case of utilization of dried shrimp for making a variety of “chutneys” in Kerala and elsewhere2.               

 

 

Phenylephrine is a used as a decongestant, to dilate the pupil, to increase blood pressure, and to relieve haemorrhoids. While marketed as a decongestant, taken by mouth at recommended doses it is of unclear benefit for hay fever. It can be taken by mouth, given by injection into a vein or muscle, or applied to the skin3.

 

Common side effects when taken by mouth or injected include nausea, headache, and anxiety. Use on hemorrhoids is generally well tolerated. Severe side effects may include a slow heart rate, intestinal ischemia, chest pain, kidney failure, and tissue death at the site of injection. It is unclear if use during pregnancy or breastfeeding is safe. Phenylephrine is a selective α1-adrenergic receptor activator which results in the constriction of both arteries and veins4.

 

Phenylephrine was patented in 1927 and came into medical use in 1938.  It is available as a generic medication. In the United Kingdom the injectable formulation costs the NHS 4 pounds a vial.  Unlike pseudoephedrine, abuse of phenylephrine is very uncommon5. Phenylephrine is used as a decongestant sold as an oral medicine or as a nasal spray. It is a common ingredient in over-the-counter decongestants in the United States. Other decongestants include oxymetazoline and pseudoephedrine.

 

Sympathomimetics are widely used as a systemic nasal decongestant in over-the-counter (OTC) common cold and flu medicines. The UK OTC Directory (2005/2006) lists 21 cough and cold medicines that contain phenylephrine (PE), 34 that contain pseudoephedrine (PDE) and one containing ephedrine6. In 1959 there were 15sympathomimetic oral medicines in use in the USA as nasal decongestants7. With the recent withdrawal of phenyl propanolamine in most countries, due to concerns about abuse as a diet aid and possible links to cerebrovascular stroke8, there are now only two systemic nasal decongestants in common use worldwide, PDEand PE. Concerns about the illicit conversion of PDE into methamphetamine9,10 have obliged pharmaceutical companies in the USA to switch PDE to PE in nasal decongestant products because of the restrictions imposed on the sale of PDE to the public. In the USA the Combat Methamphetamine Epidemic Act of 2005 bans OTC sales of medicines that contain PDE and requires purchasers of ‘behind the counter’ PDE to present photo identification and to provide personal information in a log which will be kept by the seller for at least 2years. The severe restrictions imposed on the sale of PDE in the USA may be copied by the regulatory authorities in other countries and this may lead to the loss of PDE as a common cold medicine. The efficacy and safety of PDE as a systemic nasal decongestant in syrup and tablet formulations are well documented11–14, but the efficacy of PE as a nasal decongestant has received little attention in the literature. The aim of this review was to compare the efficacy and safety of the two nasal decongestants and highlight important differences, rather than provide an in-depth review of each medicine.

 

From the preceding interpretations and also on the literature study there are flimsy reports on the isolation and characterization of bioactive compound, and also there are no reports for the past one decade on their population in relation to severe climatic changes of the present day hence the present investigation has been undertaken to present an authenticated report.

 

MATERIALS AND METHODS:

Animal Collection:

The sand crabs, Emerita asiatica (Figs. 1 and 2) were collected from Thiruvidandhai beach (Figs. 3 and 4) 13ş 06' N, 80ş 24' E, located on the Kanchipuram District, Tamil Nadu, India, 41 K.M away from Chennai, on the  East Coast Road route to Mahabalipuram. Sand crabs were collected by hand picking method.

 

Preparation of Extract:  

From the freshly collected crabs, a total of about 100 g of body muscle, was removed and was macerated. The whole muscle mass was divided into two sub samples each weighing 50g. The sample was hydrated with sufficient hydrating agent (Sodium sulphate) and the sample was refluxed in methanol for 6 hours and the extracts were used to isolate the bioactive compound by GC-MS analysis.

 

GC-MS Analysis:

Gas Chromatograph:

A Shimadzu GC-2010 Plus gas chromatograph was equipped with a straight deactivated 2 mm direct injector liner and a 15m Alltech EC-5 column (250µ I.D., 0.25µ film thickness). A split injection was used for sample introduction and the split ratio was set to 10:1. The oven temperature program was programmed to start at 35°C, hold for 2minutes, then ramp at 20°C per minute to 450°C and hold for 5 minutes. The helium carrier gas was set to 2 ml/minute flow rate (constant flow mode).

 

Mass Spectrum:

A Direct connection with capillary column metal quad pole mass filter pre rod mass spectrometer operating in electron ionization (EI) mode with software GCMS solution ver. 2.6 was used for all analyses. Low-resolution mass spectra were acquired at a resolving power of 1000 (20% height definition) and scanning from m/z 25 to m/z 1000 at 0.3 seconds per scan with a 0.2 second inter-scan delay. High resolution mass spectra were acquired at a resolving power of 5000 (20% height definition) and scanning the magnet from m/z 65 to m/z 1000 at 1 second per scan.

 

Mass spectrometry library search:

Identification of the components of the compound was matching their recorded spectra with the data bank mass spectra of NIST library V 11 provided by the instruments software. GC/MS metabolomics Database was used for the similarity search with retention index.

 

Molecular Docking Studies:

Accelrys Discovery Studio (DS) Visualizer is software used for a wide variety of different purposes, including the modelling of the binding of drugs to proteins such as enzymes and receptors. Most of the references on this Wiki to it refer to its ability to produce high-quality 3D static images of drug molecules. While it is available for free to academics and the general population it is not open-source and is not free for commercial use.

 

The three dimensional structure of Vasoconstriction protein receptor was obtained from the protein data bank (PDB ID: 1DML and 1KI2) complexed with the structure of all the bioactive compound in this study were retrieved from PubChem compound database. The raw protein from protein data bank with PDB ID 1DML and 1KI2 named Vasoconstriction protein receptor and 4C3P is further prepared for docking studies. The protein receptor was initially prepared by removing all the Heteroatoms and water molecules followed by subsequent energy minimization to remove the bad steric clashes using the software Auto dock 4.2. The 2D structures of ligand molecules were converted to 3D structures with the help of open babel.

 

Determination of binding site:

Binding and active sites of proteins are often associated with structural pockets and cavities having high affinity for candidate drugs. The catalytic site of Vasoconstriction protein receptor and phenylephrine receptor was obtained from the information available in the literature. The knowledge base information from literature and from computer program provides identification and measurements of surface accessible pockets as well as interior in accessible cavities, for proteins and other molecules. It measures analytically the area and volume of each pocket and cavity, both in solvent accessible surface and molecular surface.

 

Computation of dockings core between Cardiac stenosis protein receptor and bioactive compound was performed. All computational docking studies were carried out using Auto Dock 4.2. Automated dockings were performed to locate the appropriate binding orientations and conformations of various inhibitors in the Herpes virus protein receptor binding pocket using Auto Dock 4.2 tool according to the specified instructions. In brief, polar hydrogen atoms and Gasteiger charges were assigned to the receptor proteins. For ligands, Gasteiger partial charges were designated and non-polar hydrogen atoms were merged.  Torsions for ligands were allowed to rotate during docking procedure. The program Auto Grid was used to generate the grid maps. Each grid was cantered at the structure of the corresponding receptor. The grid dimensions were created. For all ligands, random starting positions, random orientations and torsions were used. The translation, quaternion and torsion steps were taken from default values indicated in Auto Dock 4.2. The Lamarckian genetic algorithm method was used for minimization using default parameters. Cluster analysis was performed on the docked results. The binding energy of each cluster is the mean binding energy of all the conformations present within the cluster; the cluster with lowest binding energy and higher number of conformations within it was selected as the docked pose of the particular ligand.

 

In vitro studies – Anti Microbial activity:

Sterile MHA plates were prepared, by autoclaving the medium at 121şC for 15 min. the media was distributed onto sterile Petri plates and allowed to solidify. The pure bacterial strains such as Bacillus subtilis and Pseudomonas aeruginosa were procured from the IBMS (Institute of Basic Medical Sciences, University of Madras, Taramani Campus) and these were spread on the two different plates separately. Empty discs were dipped with sterile forceps in muscle mass extract at different concentrations such as 25 µL, 50 µL, 75 µL and 100µL which was placed in a plate and the zone of inhibition were observed.

 

RESULTS AND DISCUSSION:

The results obtained on the study of isolation of bioactive compound revealed interesting facts.

 

GC-MS Analysis:

The results which have been showed through GC-MS indicated the presence 27 bioactive compounds in the muscle mass of the Sand crab, Emerita asiatica. The highest peak value 15.581 obtained between the 16th to 17th minutes (Table. 1 and Fig.1). In Line #:24 real time: 29min: 083 and Mass Peaks: 14. In the case of Gas Chromatogram Library the molecular weight 167 and retention index 1574 was obtained. The compound was called as Phenylephrine C9H13NO2 (Fig.2).

 

Molecular Docking:

The Phenylephrine which was used in this study showed the binding energies in the range -2.46 and 15.83 kcal/mol (Table. 2) which is in very good agreement with the standard and ideal binding energy. The present analysis also showed that the Phenylephrine is having better affinity.

 

So Phenylephrine is the potential lead molecule for the inhibition of Vasoconstriction protein and TRP 88, ARG 116 and 83, CYS 282, LYS 262, THR 42, ALA 144, MET 165, ILE 105 are the most important residues for potential drug target as carbon hydrogen bond, conventional hydrogen bond and Vander Waals interaction. Based on the acceptor we can conclude the affinity between Vasoconstriction protein receptor as well as Phenylephrine compound. If the inhibition constant rate is high, the action between Vasoconstriction cells and the Phenylephrine compound is strong. It will suppress the proliferation of the Vasoconstriction cells (Fig. 3, 4 and 5).

 


 

Table 1. GC-MS Analysis of Bioactive Compound Phenylephrine.

Peak Report TIC

Peak#

R. Time

Name

Area

Area%

Height

Height%

1.                  

5.085

(3-Methyl-oxiran-2-yl)-methanol

1571040

1.13

301701

1.29

2.                  

5.672

Di-alpha-L-xylofluoranose 1,2’:2,1’-dihydride

13730

0.01

10120

0.04

3.                  

7.423

1,2,3-Benzenetriol $$ Pyrogallol $$ C.I. Oxide

102553

0.07

11359

0.05

4.                  

7.608

Isosorbide $$ D-Glucitol, 1,4:3,6-dianhydro-$$

107313

0.08

30409

0.13

5.                  

7.732

Cyclohexanamine, N-3-butenyl-N-methyl-$$

101371

0.07

31716

0.14

6.                  

7.783

L-Histidine, 1-methyl-$$ Histidine, l-methyl-

74665

0.05

24760

0.11

7.                  

7.967

6-Methoxy-2-phenacyloxy-3(2H)-pyridazation

30713

0.02

4509

0.02

8.                  

8.488

Fumaric acid-2-methylpent-3-yl propyl ester

63957

0.05

21188

0.09

9.                  

8.637

Phenol,2,4-bis(1,1-dimethyl)-$$ Phenol,

132127

0.09

46335

0.20

10.                

9.516

2-Pyrazoline-3-carboxylic acid, 5-hydroxyl-1-[

64251

0.05

13289

0.06

11.                

9.583

1H-Pyrazole-4-carbonitrile, 5-amino-1-pyridine

16849

0.01

7785

0.03

12.                

10.400

Iren, teracarbonyl(pyridine)-,(tb-5-12)-$$ Tc

47331

0.03

35531

0.15

13.                

10.500

Imidazole, 2-amino-5-[2-carboxy)vinyl]-

159602

0.11

21581

0.09

14.                

13.225

Adenosine, 4’-de{hydroxymethyl}-4’-[N-ethylate]

10364

0.01

7350

0.03

15.                

14.450

3-Butenamide

21195

0.02

13835

0.06

16.                

15.000

1-Methyldecylamine $$ 2-Undecyclamine $$ 2-

40849

0.03

5991

0.03

17.                

15.581

Caffeine $$ 1H-Purine-2,6-dione, 3,7-dihydro-

135982217

97.6

22619045

96.94

18.                

16.064

1H-Pyrrole, 1-ethyl- $$ Pyrrole, 1-ethyl- $$ N

5278

0.00

3993

0.02

19.                

16.165

Theobromine $$ 1H-Purine-2,6-dione, 3,7-dih

488591

0.35

65517

0.28

20.                

16.817

Allylmalonic acid $$ Propanedioic acid, 2-pro

10248

0.01

4847

0.02

21.                

19.658

N-dl-Alanylglycine $$ Glycine, N-DL-alanyl-

25562

0.02

4839

0.02

22.                

20.336

dl-Alpha-amino-epsilon-caprolactam $$ 3-Am

47986

0.03

7611

0.03

23.                

20.850

Urea, N,N''-(2-methylpropylidene)bis- $$ Ure

45805

0.03

7111

0.03

24.                

29.083

dl-Phenylephrine $$ Benzenemethanol, 3-hydr

5863

0.00

8198

0.04

25.                

30.925

dl-Alanylglycylglycine $$ (([(2-Aminopropan

28652

0.02

8299

0.04

26.                

33.275

Hydrazine, N-(N-methyl-1-azacyclotridecan-2

31750

0.02

5619

0.02

27.                

39.188

dl-Phenylephrine $$ Benzenemethanol, 3-hydr

15662

0.01

5704

0.02

28.                

41.917

Cephaloridine $$ Pyridinium, 1-[[2-carboxy-8

11082

0.01

5814

0.02

 

 

 

139256606

100.00

23334059

100.00

 

 

Figure 1. Gas Chromatography Mass Spectrophotometry Analysis of Bioactive Compound Phenylephrine

Figure 2. Isolated bioactive compound phenylephrine at 24th line

 


Table 2. The Binding Strength of investigated Ligand with the Vasoconstriction Protein (3TO3)

Protein  Pdb ID

Ligand

 

Binding energy Inhibition

(kcal/ mol)

Constant (µm)

3TO3

Phenylephrine

-2.46

15.83

 

Figure 3. PDB ID-3TO3 (Vasoconstriction Protein)

 

Figure 4. Phenylephrine– Ligand

Polyephrine is resistant to the actions of monoamine oxidase and between 43% and 96% of an oral dose is excreted unchanged in the urine 15. In oral OTC doses, PE has minimal effects on the cardiovascular system 14, 16, 17. When administered intravenously, PE causes an increase in arterial blood pressure and bradycardia18 and may also cause coronary vasospasm. The threshold dosage of PE administered orally in man for any effects on the cardiovascular system is about 50 mg and at this dose PE causes a decline in heart rate and a slight increase in arterial blood pressure 16.

 

 

*Green: Hydrogen acceptor (Protein)

 

*Pink: Hydrogen Donor (Ligand)

Figure 5. Binding Interactions of Phenylephrine in 3TO3 Receptor

 

 

 

In vitro Studies – Antimicrobial activities:

Antimicrobial activities showed some interesting results. Klebsiella pneumoniae organism showed 7 mm zone of inhibition in 25µl, 9 mm, 12 mm, 25 mm in 50, 75 and 100 µl respectively. 10 mm was observed in control disc ampicillin (Table. 3 and Fig. 6). Staphylococcus aureus is concerned the maximum zone of inhibition 21 mm was observed in 100 µl concentration; no zone was observed in 25µl (Fig. 7).

 

The protein-ligand interaction plays a significant role in structural based drug designing. In the present work, receptor for Vasoconstriction protein receptor and bioactive compound has been taken and the potential drugs have been identified that can be used against for Vasoconstriction. By applying computational approaches, it has been tried to understand the mechanism of interactions and binding affinity between bioactive compound and Vasoconstriction protein receptor.

 

Table 3. Antimicrobial activity of Crude Extract.

S. No

Organisms

Crude Extract In Different Concentrations

25 µL

50 µL

75 µL

100 µL

Ampicillin

1

Klebsiella pneumoniae

7

9

12

25

10

2

Staphylococcus aureus

No Zone

11

17

21

12

*Zone of Inhibition in mm

 

Fig.6 Antimicrobial Activity of Crude Extract against Klebsiella pneumonia.

Zone of Inhibition

A.25µl – 7 mm; B.50µl – 9 mm; C.75µl – 12 mm; D.100µl – 25 mm;     E.  Ampicillin – 10 mm

 

There is no evidence that PE have adverse effects on the cardiovascular system in normotensive subjects PE are also reported to be safe in patients with controlled hypertension[19,20], although there are scant clinical data on the topic. Some concern has been expressed that any switch to PE may expose patients with cardiovascular disease to a medicine whose safety profile is not so well documented, but, given the long history and wide use of these nasal decongestants, this concern seems  unfounded21.

As with all sympathomimetics, PE should not be taken by patients suffering from hypertension, hyperthyroidism or heart disease because of the vasoconstrictor effects of the medicines. Similarly, patients suffering from Raynaud’s syndrome or taking medicines that inhibit monoamine oxidase (MAO) should consult their doctor before taking PE. PE may cause retention of urine in patients with prostate problems. There is no evidence that PE have adverse effects on the cardiovascular system in normotensive subjects and PE are also reported to be safe in patients with controlled hypertension 19, 20, although there are scant clinical data on the topic. Some concern has been expressed that any switch to PE may expose patients with cardiovascular disease to a medicine whose safety profile is not so well documented, but, given the long history and wide use of these nasal decongestants, this concern seems  unfounded 21.

 

In vitro Studies - Cardiomyocytes:

Cardiomyocytes studies showed the following results. In control cell line the flask shaped cells were observed. In the case of cell line treated with the concentration of 100µg/mL the cells were shrunk and gradually disintegrated. Whereas in the case of 200 µg/mL the cells were vigorously damaged (Fig. 8).

 

In the earlier studies, in the case of antimicrobial activity of the crab G. strigosus, maximum zone of inhibition was recorded in V. cholera and the lowest zone of inhibition against S. aureus. In the positive control, maximum effect was recorded against V. parahemolyticus and the lowest zone of inhibition against K. pneumoniae. Antifungal activity of the hemolymph of the crab G. strigosus against the fungal strains did not show any activity against the tested strains. Similar result was observed with the hemolymph of some brachyuran crabs against clinical pathogens which is coincided with our results 22-29. So the current study reveals the frequent consumption of sand crab may leads to the narrowing of blood vessels in the cardiac region, rare occasion consumption may be recommended.

Figure 7. Antimicrobial Activity of Crude Extract against Staphylococcus aureus.

Zone of Inhibition -A.25µl – No Zone; B.50µl – 11 mm; C.75µl – 17 mm; D.100µl - 21 mm; E. Ampicillin – 12 mm


 

                             a. Control                                                     b. 100 mg/mL                                                             c. 200 mg/mL

Figure 8. Anti-Vasoconstriction Activity of Phenylephrine against Cardiomyocytes.

 


Hence the natural compound could be used as the template for designing therapeutic lead molecules which could results into massive reductions in therapeutics development time. This study may be the subject of experimental validation and clinical trials to establish these bioactive chemicals as more potent drug for the treatment of different Vasoconstrictions. 

 

In future the ADME/T (Absorption, Distribution, Metabolism, and Excretion/Toxicity) properties of these compounds can be calculated using the commercial ADME/T tools available thus reducing the time and cost in drug discovery process. These results will be decisive factor for determining lead bioactive chemicals for further drug discovery process.

 

In conclusion, the present investigation is the first, latest and the only report on the bioactive compound of sand crab Emerita asiatica. The GC-MS analysis showed the presence of bioactive compound called Phenylephrine. In the present work, receptor for Vasoconstriction protein has been taken and the potential drugs have been identified that can be used against Vasoconstriction. The present analysis also shows that Phenylephrine is having better affinity. In vitro studies of antimicrobial activities clearly indicated that the different concentrations of Phenylephrine bioactive compound have the potential to control the bacteria such as Klebsiella pneumoniae and Staphylococcus aureus. These results will be decisive factor for determining a lead bioactive compound for further drug discovery process.

 

CONCLUSION:

In conclusion the present investigation gives a detailed report on the analysis of the bioactive compound called Phenylephrine is having better affinity with Vasoconstriction. Hence Phenylephrine is the potential lead molecule for the inhibition of Vasoconstriction protein and the most important residues for potential drug target as carbon hydrogen bond, conventional hydrogen bond and Vander Waals interaction. In vitro studies of antimicrobial activities clearly indicated that the different concentrations of Phenylephrine bioactive compound have the potential to control the bacteria such as Klebsiella pneumoniae and Staphylococcus aureus. In vitro studies of anti-vasoconstriction activities also evidently showed the different concentrations of bioactive compound Phenylephrine have the potential to control the proliferation of Vasoconstrictor cells. Reports are very flimsy on this species studied and the reports are very ancient and hence this present investigation would give newest information on the isolation of bioactive compound for the production of pharmaceutical drugs against the Vasoconstriction. These results will be decisive factor for determining a lead bioactive compound for further drug discovery process for the vasoconstriction.

 

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Received on 02.02.2019        Modified on 12.03.2019

Accepted on 08.03.2019        © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(5):2145-2152

DOI: 10.5958/0974-360X.2019.00356.1