In developing countries, where commercial farming is essential for income, food security is being compromised as a consequence of deviation from subsistence production(Reference Rajamma1). The low-income-group population has therefore come to rely more on cheap energy from foods devoid of antioxidants and other dietary factors(Reference James, Nelson and Ralph2). Developing countries have thus become the cohabitat of diseases like malnutrition and obesity(Reference Usfar, Lebenthal and Atmarita3), cancer and chronic diseases. Hence, to address this problem, we should look for foods which can help generate income as well as provide proper nutrition and food security.
In the present paper, we have studied the antioxidant content and activity of a commercially important fresh-water pearl mussel, the Lamellidens marginalis (LM; Lamarck, 1819). This species can be considered as an important food source to solve the contradiction between commercial farming and food security, as the energy- and protein-rich flesh of the mussel is a byproduct of pearl culture. Substantiated by the antioxidant content, rarely found in animal protein, the flesh can be a complete package of energy and dietary factors besides being cheap, ethnic and easy to culture.
LM is available in the densely populated food-insecure inlands of India, Bangladesh, Burma, Sri Lanka and Nepal. The edible foot portion has been reported to have ethnomedicinal usage(Reference Prabhakar and Roy4). Mussels are unique in their content of phenol proteins(Reference Padro, Gutierrez and Saez5) and have anti-inflammatory effects in experimental models and clinical studies(Reference Emelyanov, Fedoseev and Krasnoschekova6). Hence, we studied the antioxidant content of mussel and explored the effect in in vitro and in an adjuvant-induced arthritis model. The antioxidant defence system of marine mussels was reported in the perspective of pollution and heavy-metal toxicity(Reference Manduzio, Monsinjon and Galap7). However, the dietary significance of the antioxidant has so far not been reported. This information is a rare example of integration of commerce and nutrition, both for energy and disease prevention, as a perfect piece to fit in the jigsaw of integrated nutritional approach.
Materials and methods
Chemicals
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium (ABTS) salt, EDTA, reduced glutathione, metaphosphoric acid, Folin–Ciocalteau reagent, 1-chloro-2,4-dinitrobenzene, potassium ferricyanide, ferrozin, pyrogalol and sodium nitroprusside were purchased from SRL. Thiobarbituric acid, NADPH, tert-butyl hydroperoxyde, ferric chloride, Freund's complete adjuvant (FCA) and indomethacin, Trolox, O-dianisidine dihydrochloride, xylenol orange and 1,2-diamino benzene were purchased from Sigma.
Animals
Wistar strain male albino rats, about 9–12 weeks old (120 (se 10) g), were used for the in vivo experiments. The animals were collected and housed in a controlled environment (room temperature: 23 ± 2°C, relative humidity: 60 (se 5) %, 12 h day–12 h night cycle) and fed ad libitum with a balanced diet and water. All animal experiments were approved by the Departmental Animal Ethical Committee and were in accordance with the guidelines of the committee for the purpose of control and supervision of experiments on animals, Government of India.
Collection of sample and preparation of extract
Live adult fresh-water mussels were collected from the local market of Kolkata, India, and the species was identified as LM (voucher specimen no.: M26322/5) from the Mollusca Section of Zoological Survey of India, New Alipore, Kolkata, India. Aqueous extract of LM was prepared and expressed as per μg dry weight for in vitro experiments, and as per mg wet weight for in vivo experiments.
Determination of total phenol, reducing power, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium radical-scavenging activity, total antioxidant capacity, hydrogen peroxide-scavenging and metal-chelating activity
The total phenol content of the extract was determined by Folin–Ciocalteu's reagent, according to the method of Taga et al. (Reference Taga, Miller and Pratt8). The phenol content was evaluated from gallic acid standard curve (5–200 μg) and the value was expressed in terms of gallic acid equivalents.
Reducing power of the extract was determined according to Oyaizu(Reference Oyaizu9) and expressed in terms of ascorbic acid equivalent.
The ABTS∙+ radical-scavenging activity of LM was assessed according to Re et al. (Reference Re, Pellegrini and Proteggente10) and total antioxidant capacity was determined by comparing the ABTS∙+ radical-scavenging activity with trolox (0–2 mm) standard.
H2O2-scavenging activity of the extract was determined according to Nabavi et al. (Reference Nabavi, Ebrahimzadeh and Nabavi11) and expressed as percentage of H2O2 scavenged.
The Fe2+-chelating activity of the extract was determined according to Ebrahimzadeh et al. (Reference Ebrahimzadeh, Pourmorad and Bekhradnia12). The percentage inhibition of Fe2+–ferrozine complex formation by the extract was calculated according to the formula:
$$\begin{eqnarray} Inhibition\,(\%) = (Abs_{control} - Abs_{sample})/(Abs_{control})\times 100, \end{eqnarray}$$
where Abscontrol and Abssample are the absorbance of control and absorbance of sample, respectively. The metal-chelating activity of LM at the dose 100 mg/ml was expressed in terms of ascorbic acid equivalents.
Induction of oxidative stress associated with inflammatory arthritis and evaluation of oxidative stress defence system and pro-oxidant markers
Arthritis was induced in rats and the experiment was carried out as described by Chakraborty et al. (Reference Chakraborty, Bhattacharya and Bhattacharjee13). Experimental animals were divided on the next day after adjuvant injection into the following five groups, I–V, for respectively, the saline-injected normal control, FCA-injected arthritic control, arthritic animals supplemented with LM 1 (500 mg/kg per d, per oral), LM 2 (1 g/kg per d, per oral) and indomethacin (1 mg/kg per d, per oral). Treatment was given from 1 to 13 d. On the 15th day, blood was collected, and serum (as per mg of protein(Reference Lowry, Rosebrough and Farr14)) and haemolysate (as per mg of Hb) markers of oxidative stress were assessed.
Serum superoxide dismutase (SOD) activity was estimated by measuring the percentage inhibition of the pyrogalol auto-oxidation by SOD according to the standard method(Reference Marklund and Marklund15). Here, one unit of SOD was defined as the enzyme activity that inhibits the auto-oxidation of pyrogalol by 50 %. Catalase activity of haemolysate was estimated using the method of Beers & Sizer(Reference Beers and Sizer16). Serum sulphydril group content was assayed as described by Elman(Reference Elman17). Serum glutathione peroxidase (GPx) activity was estimated using the method of Paglia & Valentine(Reference Paglia and Valentine18). Serum glutathione transferase (GST) activity was estimated using the method of Habig & Jakoby(Reference Habig and Jakoby19). Serum total antioxidant status (TAS) value was estimated using the method of Re et al. (Reference Re, Pellegrini and Proteggente10), Serum lipid peroxidation level by Buege & Aust(Reference Buege and Aust20) and serum NO synthase activity by the method of Granger et al. (Reference Granger, Anstey and Miller21). Total oxidant status (TOS) was assessed using the method of Erel(Reference Erel22) and oxidative stress index, the ratio of TOS and TAS, was measured by Harma et al. (Reference Harma, Harma and Erel23) and expressed as arbitrary units. To perform the calculation, the result unit of TAS was changed to μm Trolox equivalents, and oxidative stress index value was calculated as follows: oxidative stress index = ((TOS, μm-H2O2 equivalent) × 100/ (TAS, μm-Trolox equivalents)).
Statistical methods
All the results were expressed as means with their standard errors, n 6. The level of significance was determined by one-way ANOVA followed by Tukey's post hoc test. A value of P < 0·05 was considered as significant. Pearson's correlation coefficient (r) was evaluated between total phenol content and reducing power of LM. All statistical analyses were performed using Origin 7 and MS-Office Excel 2007 software packages.
Results
Antioxidant content of Lamellidens marginalis extract
The total phenol content of LM was found to be 82·81 (se 0·75) μg gallic acid equivalent per mg of LM. Reducing power of per mg LM was comparable with 16·56 (se 1·06) μg of ascorbic acid. Correlation between phenol content and reducing power of LM (r 0·98) was found to be significant (P = 0·003).
LM showed ABTS∙+-scavenging activity and H2O2-scavenging activity dose dependently. The half maximal inhibitory concentration value of LM for ABTS∙+-scavenging activity was 7·81 mg/ml (1·299 mm-Trolox equivalents) and for H2O2-scavenging activity was 0·343 mg/ml.
LM showed 19·74 % inhibition in Fe2+–ferrozin complex formation at the dose of 100 mg/ml concentration which was found to be 7·086 μg EDTA equivalents.
In vivo antioxidant activity of Lamellidens marginalis extract in Freund's complete adjuvant-induced arthritis model after oral supplementation
LM treatment significantly restored antioxidant defence systems. SOD, total thiol (~SH), GST level in serum, and catalase concentration of haemolysate were changed significantly in the arthritis group of animals, as shown in Table 1. Serum SOD, ~SH, GST, GPx, TAS level and catalase concentration of haemolysate were found to be significantly decreased (P < 0·05) in adjuvant-injected arthritic rats when compared with normal rats after the 15th day of FCA injection. LM1, LM2 and indomethacin-treated rats showed significant increases (P < 0·05) in serum SOD, ~SH, GST, GPx level and catalase concentration on the 15th day after FCA injection when compared with arthritic rats.
Effect of Lamellidens marginalis (LM) and standard on serum antioxidant marker levels in Freund's complete adjuvant-induced arthritic animal model (Mean values with their standard errors, n 6)
PO, per oral; TBARS, thiobarbituric acid-reacting substances; MDA, malonaldehyde;~SH, total thiol; GST, glutathione transferase; GPx, glutathione peroxidase; SOD, superoxide dismutase; TAS, total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index.
* Mean values were significantly different from control (P < 0·05, one-way ANOVA).
† Mean values were significantly different from arthritis (P < 0·05, one-way ANOVA).
The pro-oxidant markers, namely, serum thiobarbituric acid-reacting substance level, nitric oxide and TOS level, were found to be significantly increased (P < 0·05) in adjuvant-injected arthritic rats when compared with normal rats after the 15th day of FCA injection. LM1, LM2 and indomethacin-treated rats showed significant decreases (P < 0·05) in serum thiobarbituric acid-reacting substances, TOS and nitric oxide level on the 15th day after FCA injection when compared with arthritic rats (Table 1).
Discussion
An epidemiological association of decreased incidence of age-related diseases in humans with diets rich in polyphenols and antioxidants(Reference Tang and Halliwell24) has made antioxidant-rich plant-based foods more valued than antioxidant-scarce animal protein(Reference Carlsen, Halvorsen and Holte25), which is a paradox in protein–energy malnutrition. Phenolic antioxidants of the Indian fresh-water pearl-producing mussel, LM, can address this problem and help in food security, as it is a more nutritious(Reference Baby, Hasan and Kabir26) alternative to meat, is cheaper, and is available as a byproduct of the cash-crop, pearl. Prabhakar & Roy(Reference Prabhakar and Roy4) showed that the foot portion of the mollusc is eaten for its ethnomedicinal benefits in North-Bihar, India. Polyphenol proteins have been reported to be secreted from the specialised phenol glands of the mussel foot(Reference Padro, Gutierrez and Saez5). Polyphenols were reported as the main antioxidants of mussels in pollution defence(Reference Moncheva, Trakhtenberg and Katrich27). Polyphenol antioxidants are found in plants and none so far has been reported in animals/meat consumed as food. These observations have led us to explore the antioxidant activity of the foot-pad of LM and to study its correlation with the phenol content of the foot-pad.
Thus, the experiments were designed to determine the total phenol content, its correlation with the free-radical-scavenging and antioxidant activity of LM extract in the in vitro system, followed by its antioxidant efficacy in the adjuvant-induced arthritis model in rats.
Table 1 shows that LM can be considered as a food rich in antioxidants with a significant correlation between phenol content and reducing powers (r 0·98; Fig. 1), indicating that the antioxidant property of LM is highly dependent on its phenol content. To study the association of this dietary antioxidant with the prevention of in vivo oxidative stress, we selected the FCA-induced inflammatory arthritis model that mimics human rheumatoid arthritis along with the feature of severe reactive oxygen species generation(Reference Kannan, Ortmann and Kmpel28). The oxidative burst takes place within the activated macrophage to combat foreign antigens in arthritis. NO is a good indicator of this reactive oxygen species generation and thus the alteration of nitric oxide level was often estimated to determine macrophage activity(Reference Hibbs, Taintor and Vavrin29). Grant et al. (Reference Grant, Cannon and Scott30) showed that elevated NO level in FCA induced arthritis. Depletion of the body's antioxidant defence reserves takes place to combat the oxidative burst within activated macrophages. SOD, catalase and ~SH are the enzymatic and non-enzymatic antioxidant defence systems of the body, markedly altered during this process. GPx regenerates the reduced glutathione from its oxidative form to sustain the antioxidant status of the body. Significantly decreased GPx level in the arthritis animal indicated the over-usage of enzyme to regenerate reduced glutathione to resist against reactive oxygen species. GST reduces organic peroxides in the presence of reduced glutathione and converts it to oxidised form. Thus, alteration of this total system indicates alteration of the body's oxidative and antioxidant status as found in the present experiment with FCA-induced animals, which was restored due to LM treatment, thereby indicating LM to have antioxidant potential.
Regression plot of total phenol content (μg gallic acid equivalents) and reducing power (μg of ascorbic acid equivalents) of Lamellidens marginalis extract (LME). Both the values were measured in same dose of LME expressed in mg dry weight (y = 0·406x − 1·287; R 2 0·961). x, Total phenol content; y, reducing power; r 2, square of Pearson's correlation coefficient.
In summary, it could be concluded that the edible portion of LM, the pearl culture byproduct, contains antioxidants, particularly the unique phenolic proteins that are effective in the prevention of arthritis, a chronic inflammatory disease. Hence, this makes LM a suitable candidate in the design of an integrated nutritional approach.
Acknowledgements
The present work was partly funded by University of Calcutta, and Council of Scientific and Industrial Research (File No.: 09/028(0831)/2010-EMRI dated 29·03·2011). M. C. and S. B. were involved in data collection, data analysis, data interpretation, literature search and manuscript preparation. R. M. was involved in data collection analysis and manuscript preparation. D. M. was involved in data collection. R. M. was also involved in the study design, data interpretation, literature search and manuscript preparation. None of the authors had any conflict of interest in connection with the present study.
