INTRODUCTION:

Among the macro fauna commonly found in mangrove forests, brachyuran crabs are one of the most important taxa with regard to both number of species, density and total biomass ^{[1 -
7]}. Most are either fiddler crabs (Family Ocypodidae, genus Uca) or sesarmid crabs (Family Grapsidae sub family Sesarminae) ^[8].

The species is diurnally active, emerging as the tide recedes ^{[9, 5, 6]}. Surface activity terminates when burrows are re-entered and plugged. Burrow plugging also prevails at night and on hot days is dry ^{[10, 11]}. Conversely, they can actively benefit from organic matter deposited on the sediment surface under enriched situations, nutrient recycling and energy flow ^{[7, 12]}.

Fiddler crabs, important play roles in many processes. They are not only the important regulators of cord grass–derived production and decomposition (bacteria and fungi), but also important to the food web, eaten by many larger predators, such as the blue crab, rails, egrets, herons and raccoons. Fiddler crabs are also avoiding burrowers. Their activity can erode or undermine marsh banks. Their burrowing and feeding affects the aeration, and hence the growth of marsh grasses. They stimulate the turnover and mineralization of important nutrients. They are also a good environmental indicator and sensitive to environmental contaminates especially insecticides.

The primary treatments for lung cancer are the same as those for most solid tumors: surgery, chemotherapy, and radiation. Their applicability and effectiveness depend on the stage at which the cancer is diagnosed, subtype classification, and genetic characteristics ^{[13, 14]}.

Although 1-year survival rates for lung cancer have improved in the past 3 decades thanks to better surgical techniques and treatments, the 5-year survival rate for all stages combined is still relatively poor at only about 16%. Patients, whose cancer is diagnosed early, before it spreads, have a 5-year survival rate of 52%; however, just 15% of lung cancers are diagnosed at this stage. Overall, the 5-year survival rate for small cell lung cancer (6%) is lower than for non-small cell lung cancer (18%) ^{[15 -19]}.

Several efforts are underway to improve outcomes in lung cancer patients. Recommendations for routine screening of current and former smokers, emphasis on personalized genomic medicine with targeted therapies, and the development of highly specific drugs focused on destroying malignant tissue while sparing healthy cells represent key opportunities in the battle against lung cancer ^[20
-24].

Symptoms of lung cancer include chest discomfort or pain, persistent cough, trouble breathing, wheezing, bloody sputum, hoarseness, loss of appetite, unexplained weight loss, fatigue, trouble swallowing, and swelling in the face and/or veins in the neck ^[19]. Individuals with SCLC sometimes exhibit other symptoms, including the syndrome of inappropriate antidiuretic hormone secretion (SIADH). (Antidiuretic hormone, or vasopressin, helps regulate reabsorption of water in the kidneys, as well as blood pressure.) Abnormal antidiuretic hormone secretion leads to excess fluid buildup in tissues ^[25].

Sometimes patients present with symptoms that are due to the cancer but are not caused by the local presence of cancer cells. The term “paraneoplastic syndrome” refers to the collection of symptoms that result from substances produced by the tumor, and occur remotely from the tumor itself. Sometimes, paraneoplastic syndromes can appear before the cancer is diagnosed and may be the presenting symptom of the cancer. These conditions include Cushing syndrome, caused by excessive cortisol levels; paraneoplastic cerebellar degeneration (a rare neurologic disorder); or Lambert-Eaton myasthenic syndrome, a rare autoimmune disorder resulting in muscle weakness in the limbs ^[26].

Risk factors for lung cancer among never smokers include exposure to secondhand smoke, hormonal and genetic factors, air pollution, radon exposure, and occupational exposure to carcinogens ^{[27, 28]}. Never smokers diagnosed with lung cancer are more likely to have adenocarcinomas. Several reports have demonstrated a better survival rate compared with smokers ^[29,
30].

Researchers have identified numerous molecular changes that drive lung cancer development, many of which have become targets of biologic drugs designed to destroy cancerous cells or arrest their growth. Several gene mutations have been associated with lung cancer, but two have been particularly well studied ^[31].

This research program was intended to identify the bioactive compound from the fiddler crab, Uca annulipes for the production of pharmaceutical drugs against the lung cancer disease.

MATERIALS AND METHODS:

Study area

The present study was carried out in Muttukadu Estuary (Fig. 1), is situated in the intertidal mud-flats on the northern bank of the Muttukadu Estuary, Kovalam, Tamil Nadu, South India. The area of the study is situated about 35 KM away from Chennai, near the mouth of Muttukadu lake (Fig. 1.1).

Animal Collection

During low tides the Fiddler crabs were collected by hand picking method during day time. All the collected crabs were bagged, labelled and stored in 70% ethanol until further analysis.

Preparation of Extract

From the freshly collected crabs, a total of about 100 g of body muscle, was removed and was macerated. The whole mass was divided into two sub samples each weighing 50g. The sample was hydrated with sufficient hydrating agent (Sodium sulphate) following the method described by Clemmen (1985) 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 quadupole 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

Bioinformatics Tools used for the 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.

Discovery Studio

BIOVIA Discoverant is an enterprise level, validation-ready system for process and quality data access, aggregation, contextualization, analysis and reporting. The system shortens time to market and maximizes profitability by enabling design of robust GMP processes and immediate visibility into process performance, quality and compliance risk, as well as improved understanding and control of process and product variability.

Open Bable Toolbox

Open Babel is a chemical toolbox designed to speak the many languages of chemical data. It's an open, collaborative project allowing anyone to search, convert, analyze, or store data from molecular modeling, chemistry, solid-state materials, biochemistry, or related areas.

Retrieval of protein and ligands from database

The three dimensional structure of Herpes virus protein receptor was obtained from the protein data bank (PDB ID: 1DML & 1KI2) complexed with the structure of all the betulin in this study were retrieved from PubChem compound database. The raw protein from protein data bank with PDB ID 1DML & 1KI2 named Herpes virus receptor and 4C3P Liver Cancer receptor 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.

Binding and active sites of proteins are often associated with structural pockets and cavities having high afﬁnity for candidate drugs. The catalytic site of herpes virus receptor and liver cancer 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 was performed between Lung cancer protein receptor and betuline.

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 lung cancer receptor binding pocket using AutoDock 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 centered 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 121ºC for 15 min. the media was distributed onto sterile Petri plates and allowed to solidify. The pure bacterial strains such as Escherichia coli 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 100 µL which was placed in a Muller Hinton Agar plate and the zone of inhibition were observed.

In vitro studies – Anti Cancer activity

Dulbecco’s Modified Eagle Medium (DMEM), trypsin, penicillin, streptomycin, dimethyl sulphoxide (DMSO), RPMI – 1640, SRB, Fetal Bovine Serum (FBS), 0.2% Chlorhexidine and Phosphate Buffered Saline (PBS) were purchased from Hi Media, Mumbai, India. All the other chemicals used in this study were purchased locally and were analytical grade.

The 3T3 cell line used in this study was obtained from the CIC Culture Collection of the University of Granada (Spain). Cells were placed under sterile conditions in 75cm2 flasks that contained 30 ml of culture medium consisting of Dulbecco modified Eagle’s medium (DMEM) + 2mM of glutamine + 10% bovine calf serum. Flasks were kept at 37°C in an atmosphere of 5% CO2 and 95% humidity until cells reached confluence. The cells were cultured and maintained for a minimum of two passages and the third passage cells were used for this study.

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 24 bioactive compounds in the muscle mass of the Fiddler crab, Uca annulipes. The highest peak value 15.912 obtained between the 15^th to 16^th minutes (Table. 1 and Fig.1). In Line #:24 real time: 45min: 633 and Mass Peaks: 218. In the case of Gas Chromatogram Library the molecular weight 442 and retention index 3090 was obtained. The compound was named as Betuline (Table. 1 and Fig.2).

Fig. 1 Gas Chromatography-Mass Spectrophotometry Analysis of Bioactive compound.

Fig. 2 Bioactive Compound Betuline in the 24th Line.

Peak Report TIC

Table. 1 GC-MS Analysis of bioactive compound.

Peak#	R.Time	Name	Area	Area%	Height	Height%
1.	5.090	(3-Methyl-oxiran-2-yl)-methanol	1853569	0.48	380973	0.88
2.	50181	2-Hydroxymethyl-2-methyl-pyrrolidine-1-cart	5915850	1.54	519920	1.21
3.	50617	2H-Azepine-2-thione, hexahydro-$$, epsilon,	1247098	0.32	95734	0.22
4.	5.827	Acetamide, N-ethyl-$$ Acetamidoethane $$ E	13498337	3.51	2113992	4.91
5.	6.200	Threitol, 2-O-decyl-	2720289	0.07	40640	0.09
6.	7.577	Sucrose $$, alpha,-D-Glucopyranoside, beta,-	437974	0.11	70877	0.16
7.	8.089	Beta-Cedren-9-alpha-ol	133674	0.03	40759	0.09
8.	8.491	Fumaric acid, 2-methylpent-3-yl propyl ester	236924	0.06	66337	0.15
9.	8.636	Phenol, 2,4-bist(1,1-dimethylethyl)-$$ Phenil	146567	0.04	55318	0.13
10.	9.878	3-Octadecene, (E)-$$ (3E)-3-Octacedene # $$	501899	0.13	166487	0.39
11.	10.077	Furan, 2-butyltetrahydro-$$ Octane, 1,4-epox	436107	0.11	131316	0.31
12.	13.577	2,3,5,6-Detetrahydrocyclohexane, 2,6-di-t-b	31569	0.01	17697	0.04
13.	15.627	Caffeine $$ 1H-Purine-2,6-dione, 3,7-dihydro	207312218	53.85	27910508	64.84
14.	15.941	1,2-Benzenedicarboxylic acid, bis(2-methylpro)	266266	0.07	92729	0.22
15.	18.550	Imidazole-5-carboxylic acid, 2-amino-	54221	0.01	12912	0.03
16.	22.357	(+)-Isomenthol	590107	0.15	148601	0.35
17.	24.156	Methyl n-hexadecyl ketone $$ 2-Octadecanone	150018	0.04	33186	0.08
18.	24.337	n-Butyl myristate $$ myristic acid n-butyl ester	218144	0.06	35975	0.08
19.	44.008	2,5-Dihydrxyacetophenone, bis(trimethylsily)	4083877	1.06	515252	1.20
20.	44.520	Chondrillasterol $$ Stigmasta-72d2-dien-3-ol,	25196785	6.54	1148578	2.67
21.	45.125	Trimethyl[4-(1,1,3,3,-tetramethylbutyl)phenoxy	52333041	13.59	1944628	4.52
22.	45.292	3-Ethoxy-1,1,1,5,5,5-hexamethyl-3-(trimethyl)	21967308	5.71	2187051	5.08
23.	45.408	Trimethyl[4-(1,1,3,3,-tetramethylbutyl)phenoxy	1485919	3.86	2348527	5.46
24.	45.633	Betulin $$ Lup-20(29)-ene-3,28-diol, (3.beta)	33245068	8.63	2970191	6.90
			385016059	100.00	43048188	100.00

Table. 2 The binding strength of investigated ligands with the Lung cancer protein (5EEM)

Protein Pdb ID	Ligand	Binding energy inhibition
		(kcal/ mol)	Constant(µm)
5EEM	Betuline	-12.3	660.33

Fig. 3 PDB ID-5EEM (Lung Cancer Protein)

Fig. 4 Betuline Ligand.

*Green Colour: Hydrogen acceptor (Protein)

*Pink Colour: Hydrogen Donor (Ligand)

Fig. 5 Binding Interactions of Betuline in 5EEM Receptor

Molecular Docking

The betuline which was used in this study showed the binding energies in the range -12.3 & -660.33 kcal/mol (Table. 2) which is in very good agreement with the standard and ideal binding energy. The present analysis also shows that the betuline is having better affinity. So betuline is the potential lead molecule for the inhibition of lung cancer protein and THR 52, ALA 164, MET 137, ILE 97, TRP 91, ARG 102 & 63, CYS 291, LYS 273 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 lung cancer protein receptor as well as betuline compound. If the inhibition constant rate is high, the action between lung cancer cells and the betuline compound is strong. It will suppress the proliferation of the cancer cells (Fig. 3, 4 and 5).

Fig.6 Antimicrobial Activity of Bioactive compound Betuline against Escherichia coli.

Zone of Inhibition

A.25µl – 4 mm; B.50µl – 7 mm; C.75µl – 12 mm; D.100µl - 24 mm; E. Ampicillin – 12 mm

Fig.7 Antimicrobial Activity of Bioactive compound Betuline against Pseudomonas aeruginosa.

Zone of Inhibition

A.25µl – 6 mm; B.50µl – 9 mm; C.75µl – 14 mm; D.100µl - 22 mm; E. Ampicillin – 8 mm

In vitro Studies – Antimicrobial activities

Antimicrobial activities showed some interesting results. Escherichia coli organism showed 4 mm zone of inhibition in 25µl, 7 mm, 12 mm, 24 mm in 50, 75 and 100 µl respectively. 12 mm was observed in control disc ampicillin (Fig. 6). Pseudomonas aeruginosa is concerned the maximum zone of inhibition 22 mm was observed in 100 µl concentration; the minimum 6 mm was observed in 25 µl (Table. 3 and Fig. 7).

a. Control

b.100 mg/mL

c. 200 mg/mL

Fig. 8 Anticancer activity of betuline against Lung cancer cell line.

In vitro Studies - Cancer Cell line Studies

Cancer cell line studies showed the following results. In control cell line the flask shaped tumor cells were observed. In the case of the cancer cell line treated with the concentration of 100 µg/mL the cells were shrunk and gradually disintergrated. The same cell lines treated with the concentration of 200 µg/mL, the tumor cells were totally damaged (Fig. 8).

The protein-ligand interaction plays a signiﬁcant role in structural based drug designing. In the present work, receptor for lung cancer protein receptor has been taken and the potential drugs have been identiﬁed that can be used against lung cancer. By applying computational approaches, it has been tried to understand the mechanism of interactions and binding afﬁnity between Betuline and lung cancer protein receptor. The betulin which was used in this study showed the binding energies in the range -12.3 & -660.33 kcal/mol, which is in very good agreement with the standard and ideal binding energy. The present analysis also shows that the betuline is having better affinity. So betuline is the potential lead molecule for the inhibition of lung cancer protein and THR 52, ALA 164, MET 137, ILE 97, TRP 91, ARG 102 & 63, CYS 291, LYS 273, are the most important residues for potential drug target as carbon hydrogen bond, conventional hydrogen bond and Vander Waals interaction. 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 betulin as more potent drug for the treatment of different cancers in general and breast cancer in particular. 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 a lead bioactive compound for further drug discovery process.

Reports are very scanty on this species studied and the reports are very old and hence this present investigation would give latest information on the isolation of bioactive compound for the production of pharmaceutical drugs against the lung cancer.

CONCLUSION:

In conclusion the present investigation gives a detailed report on the bioactive compound called betuline is having better affinity with the lung cancer cells. Hence betuline is the potential lead molecule for the inhibition of lung cancer 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 betuline bioactive compound have the potential to control the bacteria such as Escherichia coli and Pseudomonas aeruginosa. In vitro studies of anticancer activities also evidently showed the different concentrations of bioactive compound betuline have the potential to control the proliferation of Lung cancer cells. Reports are very scanty on this species studied and the reports are very old and hence this present investigation would give latest information on the isolation of bioactive compound for the production of pharmaceutical drugs against the lung cancer. These results will be decisive factor for determining a lead bioactive compound for further drug discovery process for the lung cancer.

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Received on 01.03.2019 Modified on 12.04.2019

Research J. Pharm. and Tech. 2019; 12(4): 1849-1856.

DOI: 10.5958/0974-360X.2019.00309.3