alexa Antioxidant Activity in Hard and Soft Shell Crabs of Charybdis lucifera(Fabricius, 1798) | Open Access Journals
ISSN: 2155-9546
Journal of Aquaculture Research & Development
Like us on:
Make the best use of Scientific Research and information from our 700+ peer reviewed, Open Access Journals that operates with the help of 50,000+ Editorial Board Members and esteemed reviewers and 1000+ Scientific associations in Medical, Clinical, Pharmaceutical, Engineering, Technology and Management Fields.
Meet Inspiring Speakers and Experts at our 3000+ Global Conferenceseries Events with over 600+ Conferences, 1200+ Symposiums and 1200+ Workshops on
Medical, Pharma, Engineering, Science, Technology and Business

Antioxidant Activity in Hard and Soft Shell Crabs of Charybdis lucifera(Fabricius, 1798)

Soundarapandian P*, Shyamalendu Roy and Varadharajan D
Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, Tamil Nadu, India
Corresponding Author : Soundarapandian P
Faculty of Marine Sciences
Centre of Advanced Study in Marine Biology
Annamalai University, Parangipettai, Tamil Nadu, India
Tel: 04144-243223
E-mail: [email protected]
Received August 14, 2014; Accepted October 31, 2014; Published November 10, 2014
Citation: Soundarapandian P, Roy S, Varadharajan D (2014) Antioxidant Activity in Hard and Soft Shell Crabs of Charybdis lucifera (Fabricius, 1798). J Aquac Res Development 5:288. doi:10.4172/2155-9546.1000288
Copyright: © 2014 Soundarapandian P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Related article at
DownloadPubmed DownloadScholar Google

Visit for more related articles at Journal of Aquaculture Research & Development

Abstract

The crab fishery in India is fast developing and there is a vast scope for the crab meat due to its delicacy and nutritional richness. Studies on antioxidant activity of soft shell crabs are very much need of the hour to stop the wastage of soft shell crabs and also possible utilization. Hence in the present investigation antioxidant activity is studied in soft shelled crabs which are compared with hard shell crabs of Charybdis lucifera. The soft shelled crabs showed maximum phenolic content of 48% which is tentatively higher than that of hard shelled. The total antioxidant potential of soft shelled crab exhibited maximum antioxidant potential of 49% and minimum effect of 32% was recorded in hard shelled crab. In the reducing power assay the maximum reducing ability of 59% was noticed in soft shelled crab. The lease reducing capability of 42% was recorded in hard shelled crab. A meager scavenging potential of 28% and 29% was recorded in both the crabs. In deoxyribose radical scavenging activity both the crab tissues exhibited a confined range of 30% scavenging role where the reference drug of Vitamin E records 86% of scavenging potential. In soft shelled crab was characterized with 59% DPPH free radical scavenging ability whereas the hard shelled with 48% was recorded. The results suggest that soft shelled crabs of Charybdis lucifera show antioxidant property than hard shell crabs. So the soft shelled crab may be used for the preparation of antioxidant and this will prevent the wastage of useful soft shelled crabs from the landing centers in some extend. To confirm this further extensive study is needed in this regard.

Keywords
Antioxidant activity; Soft; Hard shelled; Haemolymph; Charybdis lucifera
Introduction
The crabs rank third after shrimps and lobsters for their esteemed seafood delicacy and also the value of fishery they support. The crab fishery in India is fast developing and there is a vast scope for the crab meat due to its delicacy and nutritional richness. The crab meat contains rich amount of protein, vitamins A and D, minerals, glycogen and free amino acids. Most of the marine crabs occurring along the Indian coasts are belonging to the family portunidae. The commercially important portunid crabs found along Parangipettai coast are Scylla serrata, S. tranquebarica, Portunus sanguinolentus, P. pelagicus, Podophthalamus vigil, C. feriata, C. lucifera, C. natator, C. granulata and C. truncata [1-3]. The oxidative deterioration of fats and oils in foods are responsible for rancid odours and flavours, with a consequent decrease in nutritional quality and safety caused by the formation of secondary, potentially toxic compounds. The additions of antioxidants are required to preserve flavour and colour and to avoid vitamin destruction. Among the synthetic types, the most frequently used food preservatives are Butyrate Hydroxyanisole (BHA), Butyrate Hydroxytoluene (BHT), Propyl Gallate (PG) and Tertbutyl Hydroquinone (TBHQ). Reports revealing that BHA and BHT could be toxic and the higher manufacturing costs and lower efficiency of natural antioxidants such tocopherols together with the increasing consciousness of consumers with regard to food additive safety, created a need identifying alternative and probably safer sources of food antioxidants [4,5]. Naturally occurring antioxidant substances also need safety testing. Cautions regarding an assumption of safety of natural antioxidants have been repeatedly advised, since the fact an antioxidant comes from a natural source does not prove its assumed safety [6]. Oxidative stress is involved in the pathology of cancer, arteriosclerosis, malaria and rheumatoid arthritis and could be play a role in neurodegenerative disease and ageing processes [7-9]. The role of antioxidants has received increased attention during the past decade. However, the use of such antioxidants have potential health hazard [10]. Therefore, in recent years, interests have been developed for searching effective natural antioxidants, since they can protect human body from free radical and retard the progress of many chronic diseases. The hemolymph, in most crustaceans is a colourless fluid containing large number of cells or hemocytes and various organic and inorganic constituents. About 90% of hemolymph is water, which determines its total volume. The hemolymph serves as a bathing medium for various tissues and organs as they lack an epithelial lining of a true coelom. Therefore, the hemolymph forms the meeting place of both the raw materials required and the products of various physiological activities of the body. Since the hemolymph is not directly connected with the external environment any change in it can be taken as a measure of the physiological state of the internal environment of the intact animal.
The haemolymph of crustacean have potent antimicrobial peptide showed diverse array against several human pathogens [11] and plays a role in host defense response including self or non-self-recognition, cell to cell communication, superoxide anion activity, melanisation, phagocytosis, cytotoxicity and encapsulation [12]. In recent years, natural products from marine samples have a wide spectrum of biological activities and numerous therapeutic applications include antiviral, antibacterial, and antitumor. Cyclic and linear peptides discovered from marine animals have increased our knowledge about new potent cytotoxic, antimicrobial, ion channels specific blockers, and many other properties with novel chemical structures associated to original mechanisms of pharmacological activity [13]. There is an increasing interest in antioxidants, particularly in those of free radicals in various diseases. These pathological and clinical backgrounds have prompted to investigate novel and potent antioxidant peptides from crab which are ultimately of therapeutic use. Hence the present study was aimed to identify the antioxidant activity of homolymph from soft and hard shelled crabs of Charybdis lucifera.
Materials and Methods
Collection of haemolymph
Healthy crabs at two different stages of moulting (pre-moulting stage i.e. hard shell crab and post-moulting stage i.e. soft shell crab) were collected from the Mudasalodai landing centre (Lat. 11º 29'N and Long. 79º 46'E) near Parangipettai. Haemolymph was collected by cutting each walking legs with a fine sterile scissor. The haemolymph was collected by using a 23-guage needle and 1.0 ml syringe contained 300 μl (4°C) precooled 10% sodium citrate solution as anticoagulant in glass distilled water. To remove haemocytes from the haemolymph it was centrifuged at 2000 rpm for 15 min at 4°C. The supernatant was collected and used for the following experiments.
Antioxidant Activities
Total polyphenolic compound
Phenolic contents of crude extracts were estimated by the method of [14]. Briefly, 100 μl aliquot of sample was mixed with 2.0 ml of 2% Na2CO3 and allowed to stand for 2 minutes at room temperature. After incubation, 100 μl of 50% Folin ciocalteou’s phenol reagent was added and the reaction mixture was mixed thoroughly and allowed to stand for 30 minutes at the room temperature in the dark. Absorbance of all the sample solutions was measured at 720 nm using spectrophotometer (Shimadzu, UV-160, Japan). Phenolic contents are expressed as quercetin equivalent per gram.
Total antioxidant activity
Total antioxidant activity was measured following the method of [15,16] 7.45 ml of sulphuric acid (0.6 mM solution), 0.9942 g of sodium sulphate (28 mM solution) and 1.2359 g of ammonium molybdate (4 mM solution) were mixed together in 250 ml of distilled water and labeled as a Total Antioxidant Capacity (TAC) reagent. 300 μl of extract was dissolved in 3 ml of TAC reagent. Blank was maintained with distilled water replacing the TAC reagent. Absorbance of all sample mixtures was measured at 695 nm. Total antioxidant activity is expressed as the number of equivalents of gallic acid.
Reducing power
Reducing power of different crude extract was determined by the method prescribed by [17]. 1.0 ml of different solvent extract containing different concentration of samples was mixed with 2.5 ml of phosphate buffer (0.2 M, pH-6.6) and 2.5 ml of potassium ferric cyanide (1%). Reaction mixture was kept in a water bath at 50°C for 20 minutes. After incubation, 2.5 ml of Trichloroacetic acid (10%) was added and centrifuged at 650 rpm for 10 minutes. From the layer, 2.5 ml solution was mixed with 2.5 ml of distilled water at 0.5 ml of ferric chloride (0.1%). Absorbance of all the solution was measured at 700 nm. Increased absorbance is indicated increased reducing power.
Hydrogen peroxide radical scavenging assay
The ability haemolymph to scavenge hydrogen peroxide was determined by following the standard procedure of [17]. Hydrogen peroxide (10 mM) solution was prepared in the phosphate buffer saline (0.1 M, PH-7.4). 1 ml (0.25 mg) of the extract was rapidly mixed with 2 ml of hydrogen peroxide solution. The absorbance was measured at 230 nm in the UV spectrophotometer (Shimazdu, UV-160) against a blank (without hydrogen peroxide) after 10 minutes of incubation at 37°C. The percentage of scavenging of hydrogen peroxide was calculated using the following formula.
Where A0-Absorbance of control
A1-Absorbance of sample
Deoxyribose radical scavenging activity
Deoxyribose non-site specific hydroxyl radical scavenging activity of solvent extract and fractions were determined according to the method of [18]. Briefly, 2.0 ml of aliquots of sample were added to the test tube containing reaction mixture of 2.0 ml of FeSO4. 7H2O (10 mm), 0.2 ml EDTA of (10 mM) and 2.0 ml of deoxyribose (10 mM). The volume was made upto 4.8 ml with phosphate buffer (0.1 M, pH 7.4) and to that 0.2 ml of H2O2 (10 mM) was added. The mixture was incubated at 37°C under dark for 4 h. After incubation, 1 ml of TCA (2.8%) and TBA (1%) were added to the mixture, and then left to stand under boiling water bath for 10 minutes. After treatment absorbance was measured at 532 nm. If the mixture was turbid, the absorbance was measured after filtration. Scavenging activity (%) was calculated using the equation given by [19].
DPPH free radicals scavenging activity assay
The scavenging activity for DPPH free radicals was measured according to the method of Shimada et al. [20]. DPPH solution was prepared at the concentration of 0.1 ml in ethanol. During the assay, the 1 ml of test solution (concentration of 0.5-3.5 mg/ml) was mixed with 1 ml DPPH solution. The mixture was incubated in dark place for 30 min at 25°C. After standing for 30 minutes, absorbance was recorded at 517 nm by UV Beckman spectrophotometer (Beckman Coulter). The percentage of DPPH free radicals scavenging activity was calculated by following equation:
Where, A0 is the absorbance of sample and A1 is the absorbance of blank.
Results
Total polyphenolic compound
The soft shelled crabs showed maximum phenolic content of 48% which is tentatively higher than that of hard shelled. However the phenolic content based antioxidant role is imperatively higher in the case of the quercetin which is used as standard drug. The blank value of reagent alone acts as a control group in this assay (Table 1).
Total antioxidant activity l
The total antioxidant potential of soft shelled crab exhibited maximum antioxidant potential of 49% and minimum effect of 32% was recorded in hard shelled crab. Gallic acid reports to have 86% of total antioxidant efficacy (Table 2).
Reducing power
In the reducing power assay the maximum reducing ability of reference drug Ascorbic acid (91%) was a dominating agent which is followed by the 59% reducing ability in soft shelled crab. The lease reducing capability of 42% was recorded in hard shelled crab (Table 3).
Hydrogen peroxide radical scavenging assay
There is no significant difference of scavenging ability of H2O2 in the crab tissues. A meager scavenging potential of 28% and 29% was recorded in both the crabs. Ascorbic acid was resulted with 79% scavenging potential (Table 4).
Deoxyribose radical scavenging activity
In Deoxyribose radical scavenging activity both the crab tissues exhibited a confined range of 30% scavenging role where the reference drug of vitamin E records 86% of scavenging potential. The free radical scavenging potential of both the crabs was imperatively very low in accordance with the standard drug (Table 5).
DPPH free radicals scavenging activity assay
In DPPH radical scavenging activity the crab tissues revealed a considerable scavenging role. In soft shelled crab was characterized with 59% DPPH free radical scavenging ability whereas the hard shelled with 48% was recorded. The reference drug of Gallic acid tends to have 90% scavenging ability (Table 6).
Discussion
Antioxdant activity is fundamental property and much important for life. Many of the biological functions such as anti-mutagenicity, anti-carcinogenicity and anti-aging, among others originate from this property [21]. Literature survey revealed that shellfish waste is rich source of phenolic compounds. Phenolic compounds play an important role in the antioxidative properties and phenolic substances were also reported to possess a wide range of biological effects, including antioxidant, antimicrobial, anti-inflammatory and anticancer activity [22]. In the present study the soft shelled crab showed maximum phenolic content of 48% which is tentatively higher than that of hard shelled crab. The soft shelled crab exhibited maximum antioxidant potential of 49% and minimum effect of 32% was recorded in hard shelled crab. Sudhakar [23] recorded the total antioxidant activity ranged from 28.52% to 80.26% at varying concentrations (0.5 to 10 mg/ ml) in P. sanguinolentus crab shell chitosan sample.
In the reducing power assay the maximum reducing ability of reference drug Ascorbic acid (91%) was a dominating agent which is followed by the 59% reducing ability in soft shelled crab. The lease reducing capability of 42% was recorded in hard shelled crab. The reducing power of chitosan ranged from P. sanguinolentus chitosan, 0.20% to 0.42% at varying concentrations (0.5 to 10 mg/ml) [23]. In hydrogen peroxide activity there is no significant difference between hard and soft shelled crabs. A meager scavenging potential of 28% and 29% was recorded in both the crabs. Ascorbic acid was resulted with 79% scavenging potential. Scavenging abilities on hydroxyl radicals were 20.05% at varying 0.5 mg/ml concentration of chitin [23].
In Deoxyribose radical scavenging activity both the crab tissues exhibited a confined range of 30% scavenging role where the reference drug of Vitamin E records 86% of scavenging potential. The free radical scavenging potential of both the crabs was imperatively very low in accordance with the standard drug. In DPPH radical scavenging activity the crab tissues revealed a considerable scavenging role. The soft shelled was characterized with 59% DPPH free radical scavenging ability were of the hard shelled with 48% was recorded. The reference drug of Gallic acid tends to have 90% scavenging ability. DPPH radicals were reported at 41.35% at 10 mg/ml [23].
Lin et al. [24] found that the N-alkylated disaccharide chitosan derivatives with different Degrees of Substitution (DS) of 20-30% exhibited the highest DPPH radical scavenging abilities of 80-95% at 0.1 mg/ml, followed by the derivatives with DS of 40-50% and 60-70%. Apparently, the scavenging ability of chitosan might be reduced after sulfation or might be enhanced after N-alkylation of disaccharide. Oktay et al. [25] suggested that free amino group in the -CH2CH2NH2 plays an important role in the free radical scavenging activity. In addition to this, cytotoxic effect of AEC was also assessed using human lung fibroblast (MRC-5) cell line and AEC showed less toxic against MRC-5.
A free radical can be defined as any species containing one or more unpaired electron, includes hydrogen atom, Tricholomethyl, Super oxide, Hydroxyl, Thioyl/perthiyl (RS/RSS), Peroxyl, Alkoxyl (RO2, RO), oxides of nitrogen (NO, NO2 ), nitrogen-centered radicals (C6H5N=N) and transition metal ions (Fe, Cu etc) [26]. Superoxide anion (O2 -) is considered as the primary ROS, and further interacts with other molecules to generate secondary ROS, either directly or prevalently through enzyme or metal catalysed processes [27]. Hydroxyl radical can be generated in biologically relevant systems by multiple reactions. A mixture of H2O2 with Fe2+ salt oxidizes many different organic molecules. Hydroxyl radicals are responsible for a large part of the damage done to cellular DNA, proteins and lipids by ionizing radiation. DNA damage, especially double-strand breaks cannot easily be repaired by the cell. Oxygen normally present in most biological systems aggravates the damage done by ionizing radiation.
Superoxide is far less reactive with non-radical species in aqueous solution. It does not react quickly with some other radicals such as NO; certain iron-sulphur clusters in enzymes and certain phenoxyl radicals. Peroxyl (RO2) and alkoxyl (RO) radicals are good oxidizing agents. Radicals (RO) formed in biological systems often undergo rapid molecular rearrangement to other radicals species. For example: RO2, Radicals oxidize ascorbate and NADH, later leading toO2 - formation in the presence of O2. In-vitro, thiols (especially reduced glutathions GSH) are often regarded as antioxidant agents, since them potent protein- SH groups against oxidation and can scavenge oxygen radicals and some other reactive species such as hypochlorous acid and themselves generate free radicals [26]. Kobayashi et al. [28] suggest that low molecular chitosans may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patient’s plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia. Dai et al. [29] concluded that antioxidative activates were found in the culture supernatant of Serratia italica TKU013 with squid pen as the sole carbon/ nitrogen source and the 4th day supernatant showed the strongest antioxidant activities and the highest total phenolic content. Dharmishtha et al. [30] concluded that chito oligosaccharides low molecular weight can be used as antioxidant in biological systems. All the tested compounds reduced either the hemolytic and DNA damage by inhibiting H2O2 and AAPH- radicals. Park et al. [10] reported that the scavenging activity was dependent on the number of amino groups available (degree of acetylation). Dai et al. [29] tested the efficacy of chito oligosaccharides low molecular weight as inhibitors of H2O2 and AAPH induced RBC hemolysis. Hydrogen peroxide crosses the RBC membrane and is known to react with Hb, generating highly reactive radical species including hydroxyl radicals. It has been reported that shrimp shell waste contains natural antioxidants; mainly phenolic compounds. The reducing power could be attributed mainly to the bioactive compounds associated with antioxidant activity [19,24,31]. The data provide a useful example of utilizing squid pen bio-waste material as a valuable functional ingredient. The findings of the current report appear useful for further research aiming to isolate and identify the specific compounds responsible for the antioxidant activity of TKU013-fermented supernatant. It was reported that the antioxidant activity is concomitant with the reducing power, which may serve as a significant indicator of the potential antioxidant activity for a compound [32]. Vitamin C has a protective function against oxidative damage and a powerful quencher of singlet oxygen (O2), hydroxyl (˙OH) and peroxyl (RO2) radicals. The occurrences of carotenoids in crustaceans are mainly due to the absorption of pigments from the diet, which they deposit as such or transfer metabolically to keto or hydroxyl derivatives [33,34].
One of the important characteristic of carotenoids is their ability to act as antioxidants, thus protecting cells and tissues from the damaging effect of free radicals and singlet oxygen [35]. Among the carotenoids, astaxanthin was found to be more effective than β-carotene as an antioxidant. Hydroxyl radicals were generated by direct addition of Fe2+ to a reaction mixture containing phosphate buffer under normal conditions [36]. Free radicals produced by radiation, chemical reactions and several redox reactions of various compounds may contribute to protein oxidation, DNA damage, lipid peroxidation in living tissues and cells [37]. This oxidative stress may be related to many disorders such as cancer, atherosclerosis, diabetes and liver cirrhosis [36,38].
In foods, antioxidants are added to minimize changes in flavour, aroma, color or nutritional value. Antioxidants can protect the body against damages caused by free radicals and degenerative diseases. Synthetic antioxidants usually employed in industry are effective and stable, but their use is limited in many countries because they are not considered complete safe for human health [39]. Incorporation of antioxidants into packaging materials have become popular since oxidation is a major problem affecting the food quality. Currently, the most frequently used antioxidants in active packaging are Butylated Hydroxysanisol (BHA) and Butylated Hydroxytoluene (BHT). Although these synthetic antioxidants can effectively be used in active food packing because of their high stability, low cost and efficiency, there are significant concerns related to their toxicological aspects. Moreover, uses of synthetic antioxidants are under strict regulation due to the potential health risk caused by such compounds. Therefore, extensive research has been conducted to employ some natural antioxidants such as phenolic compounds as alternatives to synthetic antioxidants [40,41]. The results suggest that soft shelled crabs of C. lucifera show antioxidant property than hard shell crabs. So the soft shelled crab may be used for the preparation of antioxidant and this will prevent the wastage of useful soft shelled crabs from the landing centers in some extend. To confirm this further extensive study is needed in this regard.
References









































Tables and Figures at a glance

Table icon Table icon Table icon
Table 1 Table 2 Table 3
Table icon Table icon Table icon
Table 4 Table 5 Table 6
Select your language of interest to view the total content in your interested language
Post your comment

Share This Article

Recommended Conferences

Article Usage

  • Total views: 11616
  • [From(publication date):
    December-2014 - Jun 26, 2017]
  • Breakdown by view type
  • HTML page views : 7837
  • PDF downloads :3779
 

Post your comment

captcha   Reload  Can't read the image? click here to refresh

Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords