Biochemistry & Nutrition

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Authentication of two bio-active fish oils by qualitative lipid profiling using semi-targeted approach: an exploratory study
(2020) Chatterjee, N.S.; Singh, A.; Vishnu, K.V.; Ajeeshkumar, K.K.; Anandan, R.; Kumar, K.A.; Mathew, S.
Authentication of Two Bio-Active Fish Oils by Qualitative Lipid Profiling Using Semi-Targeted Approach: An Exploratory Study
(Journal of AOAC International, 2020) S. Chatterjee, Niladri; Singh, Akanksha; Vishnu, K .V.; Ajeeshkumar, K.K.; Anandan, R.; Ashok Kumar, K.; Mathew, Suseela
Background: Fish oils, which are rich in health-promoting polyunsaturated fatty acids (PUFA), have emerged as promising functional foods in the global health and wellness food market. Their source regarding the fish type, season, and location of harvesting might influence the nutritional value of such bioactive oils and determine their market price. The differences in price among such oils often lead to economically motivated mislabeling and adulteration. Objective: In this study, our objective was to demonstrate how a qualitative targeted shotgun lipid profile workflow using an electrospray ionization-quadrupole-linear ion trap MS (QTrap) could differentiate fish oils originating from two different species. Methods: Five samples each of sardine (Sardinella longiceps) oil and shark (Echinorhinus brucus) liver oil were diluted to a concentration of 80 µg/mL in chloroform-methanol (1 + 2, v/v) with 5 mM ammonium acetate. These samples were directly infused into a QTrap MS. The data were acquired for 23 precursor ion and 4 neutral loss scan experiments in the positive ionization mode and compared. Results: We identified the following major lipid classes: cholesteryl ester, diacyl glycerol, triacylglycerol, monoalkyldiacylglycerol, and phophatydyl choline. The relative peak areas of the identified lipid species, when subjected to supervised multivariate analysis, could effectively distinguish the sardine oil and shark liver oil. Conclusions: The approach will be useful in establishing authenticity of fish oil and to support the regulatory agencies in dispute resolution. It can also be extended to establish authenticity in other agricultural and food commodities. Highlights: This paper reports a proof of concept for authenticating PUFA-rich fish supplements. A shotgun targeted lipidomics profile and chemometrics modeling successfully discriminated sardine oil and shark liver oil.
Biochemical characterization of liver oil of echinorhinus brucus (bramble shark) and its cytotoxic effect on neuroblastoma cell lines (SHSY-5Y)
(2016) Vishnu, K.V.; Ajeeshkumar, K.K.; Chatterjee, N.S.; Suvanish Kumar; Shyni, K.; Jayarani, R.; Suseela Mathew
The objective of the present study was to characterize the liver oil extracted from the deep sea shark, Echinorhinus brucus, caught from Central Indian Ocean and to evaluate its cytotoxic effect on neuroblastoma cell line (SHSY-5Y). Characterization of liver oil of Echinorhinus brucus revealed the presence of palmitic acid (15%), oleic acid (12%), stearic acid (8%), docosahexaenoic acid (DHA) (18%), and eicosapentaenoic acid (EPA) (16%). It was also found to be a good source of squalene (38.5%) and fat soluble vitamins such as A, D, and K (vitamin A: 17.08mg/100 g of oil, vitamin D: 15.04mg/100 g oil, and vitamin K: 11.45mg/100 g oil). Since it was found to be rich in essential fatty acids, fat soluble vitamins, and squalene, it can be considered as better dietary supplement.The oil of Echinorhinus brucus also showed high in vitro cytotoxic effect against the human neuroblastoma cell line (SHSY-5Y) and the IC50 value laid between 35 and 45 ng.
Biochemical Composition and Heavy Metal Content of Selected Marine Fish from the Gulf of Mannar, India
(Central Institute of Fisheries Technology, Cochin, India, 2015) Ajeeshkumar, K.K.; Visnu, K.V.; Remyakumari, K.R.; Navaneethan, R.; Asha, K.K.; Ganesan, B.; Chatterjee, N.S.; Anandan, R.; Suseela Mathew
Biochemical composition of five marine fish; grey bamboo shark, yellow margin trigger fish, paleedged stingray, laced moray and Japanese leatherjacket fish from the Gulf of Mannar, India were evaluated. Moisture, protein, lipid and ash content for the samples ranged between 75.07-76.74, 17.99-20.97, 1-3.5 and 1.46-1.93% respectively. Profiles of heavy metal, fatty acid and amino acid data were also collected. The predominant fatty acids recorded in all five species were docosahexaenoic acid (DHA), arachidonic acid (AA) and palmitic acid. All five species contain high content of different essential and non-essential amino acids namely glutamic acid, aspartic acid, arginine, serine and lysine. Significant amount of iron (13-50 ppm), zinc (2.5-10.5 ppm) and copper (0.18-4.03 ppm) were found in all the fishes. Among heavy metals, cadmium (0.05-0.1 ppm) was detected which is below permissible limit.
Biochemical profile and nutritional quality of Indian squid, Uroteuthis duvauceli
(2018) Remyakumari, K.R.; Ginson, J.; Ajeeshkumar, K.K.; Vishnu, K.V.; Asha, K.K.; Mathew, S.
This study was designed to find out the biochemical and nutritional profiling of Indian squid. Proximate composition of Uroteuthis duvauceli showed a content of 80.47% for moisture, 17.5% for protein, 0.52% for fat and 1.13% for ash, respectively. Amino acid analysis showed a higher content of glutamine, followed by aspartine, tryptopan, leucine, alanine and glycine. Higher concentrations of total amino acid (TAA), total essential amino acid (TEAA), total acidic amino acid (TAAA), total neutral amino acid (TNAA), total sulphur amino acid (TSAA) and total aromatic amino acid (TArAA) were observed. In case of fatty acids, saturated fatty acids, palmitic and stearic acid contributed highest quantity; whereas, DHA, EPA and arachidonic acid were the major unsaturated fatty acids in the sample. Among the macro minerals, potassium showed highest content followed by sodium and calcium. As in the case of micro minerals, magnesium content showed highest proportion and copper showed least quantity. Commendable quantities of biochemical and nutritional content in U. duvauceli signify the appropriateness of this moderately exploited resource as an essential nutrients for nutritionally deprived population.
Gastroprotective effect of Sardine oil(Sardinella longiceps) against HCL/ethanol induced ulceration in Wistar rats
(IIJFAS, 2017) Vishnu, K.V.; Ajeeshkumar, K.K.; Remyakumari, K.R.; Ganesan, B.; Niladri, S.C.; Lekshmi, R.G.K.; Shyni, K.; Mathew, S.
In vitro anti-cervical cancer effect of proteoglycans isolated from deep sea shark cartilage by inducing apoptosis and cell cycle arrest
(2017) Ajeeshkumar, K.K.; Vishnu, K.V.; Asha, K.K.; Remya Kumari, K.R.; Reshma, J.; Suseela Mathew
The present study was aimed at studying in vitro anti-cervical cancer effect of proteoglycans (PGs) isolated from deep sea shark. Anti-proliferative activity and apoptotic involvement were confirmed by following assays viz, the cell viability assay by MTT assay, morphological analysis and colony formation assay. The sample showed a significant cytotoxic activity for HeLa cells and results showed that the PGs at a concentration of 250μg/mL inhibit the colony formation capacity. Apoptosis induction was confirmed by the chromatin condensation assay by Hoechst 33342 nuclear staining. A number of apoptotic HeLa cells displayed a round and shrunken cell body and chromatin condensation inside the nucleus suggesting that PGs-induced apoptosis caused cell death. Apoptosis of HeLa cells was further verified by annexin V-FITC (fluorescein isothiocyanate) and PI (propidium iodide) staining by flow cytometry. PGs (250 μg/ml) were found to induce apoptosis and the ratio of apoptotic cells were 30.5%. Cell cycle arrest at G1/M and G2/M phase confirmed the cell death due to cell cycle arrest. Our study determined that proteoglycans effectively induced apoptosis and caused cell cycle arrestestablishing their anti-proliferative effect against cervical cancer.
Microencapsulation of fish oil - current perspectives
(ICAR - Central Institute of Fisheries Technology, Cochin, 2016) Kumar, L.R.G.; Vishnu, K.V.; Tejpal, C.S.; Ajeeshkumar, K.K.; Chatterjee, N.S.; Mathew, S.
Microencapsulation of sardine oil: Application of vanillic acid grafted chitosan as a bio-functional wall material
(2017) Vishnu, K.V.; Chatterjee, N.S.; Ajeeshkumar, K.K.; Lekshmi, R.G.K.; Tejpal, C.S.; Mathew, S.; Ravishankar, C.N.
Microencapsulation of β-carotene with vanillic acid grafted chitosan improves stability and glutathione content in rats
(2018) Asha, K.K.; Ajeeshkumar, K.K.; Chatterjee, N.S.; Anandan, R.; Mathew, S.
Protective Effects of Echinorhinus brucus Liver Oil against Induced Inflammation and Ulceration in Rats
(Central Institute of Fisheries Technology, Cochin, India, 2015) Vishnu, K.V.; Ajeeshkumar, K.K.; Asha, K.K.; Remyakumari, K.R.; Ganesan, B.; Anandan, R.; Chatterjee, N.S.; Suseela Mathew
Anti-inflammatory and anti-ulcer activities of Bramble shark (Echinorhinus brucus) liver oil were examined in rats. The oil showed significant proportion of n-3 polyunsaturated fatty acids (PUFAs), the percentages of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) being 16 and 18% respectively. The study also revealed that liver oil had a very favourable n3:n6 ratio of 4.7. Oral administration of shark liver oil at 1g kg-1 concentration significantly attenuated the formalin-induced paw edema in experimental rats. It exerted potent anti-ulcer effect against acid-ethanol mixturemediated lesion formation in the rat gastric mucosa.
Proteoglycans and its anti-cancer potentials - an overview
(ICAR - Central Institute of Fisheries Technology, Cochin, 2016) Ajeeshkumar, K.K.; Vishnu, K.V.; Asha, K.K.; Remyakumari, K.R.; Navaneethan, R.; Shyni, K.; Mathew, S.
Proteoglycans isolated from the bramble shark cartilage show potential anti-osteoarthritic properties
(2019) Ajeeshkumar, K.K.; Vishnu, K.V.; Navneethan, R.; Raj, K.; Remyakumari, K.R.; Swaminathan, T.R.; Mathew, S.; Asha, K.K.; Sreekanth, G.P.
Osteoarthritis (OA) causes articular cartilage destruction, initiating pain and inflammation in the joints, resulting in joint disability. Medications are available to manage these symptoms; however, their effects on the disease progression are limited. Loss of proteoglycans (PGs) was reported to contribute articular cartilage destruction in OA. Therapeutics approaches were previously studied in the animal models of OA. In the present study, we investigated the oral efficacy of four dosages of PGs (25 mg/kg, 50 mg/kg, 100 mg/kg and 200 mg/kg), isolated from the bramble shark cartilage, in an animal model of OA. Indomethacin was used as a bioequivalent formulation. Primarily, the mass spectrum analysis of the purified PGs obtained from bramble shark cartilage revealed the presence of two unique peptides including AGWLSDGSVR and LDGNPINLSK, that showed sequence similarity with aggrecan core-protein and epiphycan, respectively. The levels of C-reactive protein and uric acid in the OA rats were reduced when treated with PGs. Histopathology analysis displayed less cartilage erosion and neovascularization in OA rats treated with PGs. The X-ray imaging presented higher bone density with 200 mg/kg dosage of PG treatment in OA rats. The expressions of the inflammatory modulators including TNF-α, IL-1β, MMP13, NOS2, IL-10 and COX-2 were found to be moderated with PG treatment. In addition, PG treatment maintained the activities of antioxidant enzymes, including SOD and catalase in the joint tissues with a higher GSH content, in a dose-dependent manner. Taken together, our preliminary findings report the anti-osteoarthritic properties of PGs and recommend to evaluate its efficacy and safety in randomized trials.
Report on the occurrence of abnormal four-armed red-knobbed starfish protoreaster linckii (Echinodermata: Astroidea), Tuticorin coast, south-east coast of India
(Marine Biological Association of the United Kingdom, 2015) Chelladurai, G.; Balakrishnan, S.; Jayanthi, G.; Ajeeshkumar, K.K.; Mohanraj, J.
Starfish are echinoderms that live among corals and occur from the supra-littoral to the hadal zone. Deviation from pentamerism is a rare phenomenon in starfish and was observed in the red-knobbed starfish (Protoreaster linckii) collected from the Tuticorin coast, India. Eighty-five specimens were collected and, among these, one was found to be abnormal and had only four arms. The reasons for this abnormality may be due to the changes that occur during larval metamorphosis or caused by an abnormal regeneration of the arm.
Sardine oil loaded vanillic acid grafted chitosan microparticles, a new functional food ingredient: attenuates myocardial oxidative stress and apoptosis in cardiomyoblast cell lines (H9c2)
(2017) Vishnu, K.V.; Ajeeshkumar, K.K.; Chatterjee, N.S.; Lekshmi, R.G.K.; Sreerekha, P.R.; Mathew, S.; Ravishankar, C.N.
Fish oil has been widely recognized as an excellent dietary source of polyunsaturated n-3 fatty acids such as EPA and DHA. However, it can undergo oxidation easily resulting in the formation of toxic off flavor compounds such as hydro- peroxides. These compounds adversely affect the nutritional quality and may induce several stress reactions in body. To solve this problem, a new antioxidant bio-material, vanillic acid-grafted chitosan (Va-g-Ch), was synthesized and used as a wall material for microencapsulation of fish oil. The sar- dine oil loaded Va-g-Ch microparticles could be a potential functional food ingredient considering the numerous health benefits of fish oil, chitosan, and vanillic acid. The current study aimed to investigate the possible protective effect of sardine oil-loaded Va-g-Ch microparticles against doxorubicin-induced cardiotoxicity and the underlying mech- anisms. In vitro cytotoxicity evaluation was conducted using H9c2 cardiomyocytes. MTT assay revealed that effective cytoprotective effect was induced by a sample concentration of 12.5 μg/mL. Results of apoptosis by double fluorescent staining with acridine orange/ethidium bromide and caspase- 3 evaluation by ELISA substantiated the above findings. Further, flow cytometric determination of membrane poten- tial, relative expression of NF-κB by PCR, and ROS determi- nation using DCFH-DA also confirmed the protective effect of encapsulated sardine oil against doxorubicin-induced cardiotoxicity. NF-κB expression was down-regulated nearly by 50% on cells treated with encapsulated sardine oil. Altogether, the results revealed that sardine oil-loaded Va-g- Ch microparticles demonstrated potential cell protection against doxorubicin-induced oxidative stress
Screening natural content of water soluble b vitamins in fish: enzymatic extraction, HILIC separation and tandem mass spectrometric determination
(2017) Chatterjee, N.S.; Ashok Kumar, K.; Ajeeshkumar, K.K.; Remya Kumari, K.R.; Vishnu, K.V.; Anandan, R.; Suseela Mathew; Ravishankar, C.N.
Despite the potential of LC with tandem MS (MS/MS) in improving sensitivity and selectivity, analytical methods are scarce for the determination of protein-bound and phosphorylated forms of B vitamins in food. This prompted us to develop a method for LC-MS/MS determination of naturally occurring nicotinamide, nicotinic acid, thiamine, pyridoxine, riboflavin, pantothenic acid, biotin, folic acid, and cyanocobalamin in fish. Baseline separation of the vitamins was achieved in a hydrophilic interaction LC condition. An ultrasonication-assisted enzymatic extraction protocol for sample preparation was optimized and validated. The time required for extraction was significantly reduced (to 4 h), while maintaining good extraction efficiency. Acetonitrile content (80%, v/v) in the prepared sample was found to be optimum for excellent peak shape and sensitivity. The dynamic linear range of the vitamins ranged from 2.5 to 500 ng/g, and the regression coefficient values were greater than 0.99. LOQ values ranged from 0.4 to 50 ng/g for the different vitamins. The spike recovery values at 50 and 100 ng/g ranged from 87.5 to 97.5%. The intra- and interday precision values were satisfactory. Accuracy of the developed method was determined by analysis of a Certified Reference Material. The method could also be used for unambiguous determination of the natural content of the target vitamins in fish.
Screening Natural Content of Water-Soluble B Vitamins in Fish: Enzymatic Extraction, HILIC Separation, and Tandem Mass Spectrometric Determination
(Journal of AOAC International, 2017) Sekhar Chatterjee, Niladri; Ashok Kumar, K.; Ajeeshkumar, K.K.; Remya Kumari, K.R.; Vishnu, K.V.; Anandan, Rangasamy; Mathew, Suseela; Ravishankar, C.N.
Despite the potential of LC with tandem MS (MS/MS) in improving sensitivity and selectivity, analytical methods are scarce for the determination of protein-bound and phosphorylated forms of B vitamins in food. This prompted us to develop a method for LC-MS/MS determination of naturally occurring nicotinamide, nicotinic acid, thiamine, pyridoxine, riboflavin, pantothenic acid, biotin, folic acid, and cyanocobalamin in fish. Baseline separation of the vitamins was achieved in a hydrophilic interaction LC condition. An ultrasonication-assisted enzymatic extraction protocol for sample preparation was optimized and validated. The time required for extraction was significantly reduced (to 4 h), while maintaining good extraction efficiency. Acetonitrile content (80%, v/v) in the prepared sample was found to be optimum for excellent peak shape and sensitivity. The dynamic linear range of the vitamins ranged from 2.5 to 500 ng/g, and the regression coefficient values were greater than 0.99. LOQ values ranged from 0.4 to 50 ng/g for the different vitamins. The spike recovery values at 50 and 100 ng/g ranged from 87.5 to 97.5%. The intra- and interday precision values were satisfactory. Accuracy of the developed method was determined by analysis of a Certified Reference Material. The method could also be used for unambiguous determination of the natural content of the target vitamins in fish.

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