The narrow-clawed crayfish, formerly
Antioxidants can be defined as molecules that have the ability to prevent oxidising chain reactions caused by reactive free radicals from starting or spreading and interact with them prior to bioactive molecules of vital importance in living metabolism being affected (Ismail et al. 2004; Sirivibulkovit et al. 2018). Antioxidants can inhibit free radical reactivity through a variety of mechanisms, including hydrogen support, singlet oxygen quenching, and radical scavenging capacity (Sirivibulkovit et al. 2018; Antolovich et al. 2022). Antioxidants play an important role as a protective factor for human health. Scientific research and associated evidence indicate that antioxidants tend to reduce the risk of numerous chronic diseases, especially cancer and heart disease (Tailor & Goyal 2014). The method of 2.2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging is used as a fast simple and inexpensive process to calculate the antioxidant capacity of foods (Kirtikar & Basu 2006; Tailor & Goyal 2014).
Carotenoids are one of the classes of organic compounds widely used in the food industry for their colouring power and growth potential (Mezzomo & Ferreira 2016). Carotenoids are known as tetraterpene pigments, which are usually coloured yellow, orange and red (Maoka 2020). Commonly found in nature, carotenoids are found in almost all colourful fruits and vegetables/plants. Carotenoids currently used in industries can be chemically synthesised by extracting only a small percentage of those taken from plants or algae. Considering that consumers’ preferences and demands for natural compounds are high, it can be said that there is a general tendency towards products with natural ingredients (Mezzomo & Ferreira 2016). Photosynthetic bacteria can be found in some species of fungi, algae, plants and animals. Despite their general skeleton of 40 carbons, carotenoids are composed of eight units of isoprene. Their structures consist of a polyene chain with nine conjugated double bonds and final groups on both ends (Maoka 2020). Aquatic organisms may contain different types of carotenoids, which can be obtained nutritionally from algae and other different organisms and can be modified by metabolic reactions. Most of the carotenoids found in aquatic organisms can be cited as β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxantin and astaxanthin (Matsuno 2001; Maoka 2009, 2011, 2020).
This paper is the first attempt to report the carotenoids and their radical scavenging properties of
The present study was carried out in Atikhisar Reservoir (Çanakkale, Türkiye) (Figure 1) which is constructed on the Sarıçay Stream. Atikhisar Reservoir provides drinking water for the local people inhabiting Çanakkale (Kale 2019) and is the only source of potable water for the people in the region (Kale & Acarlı 2019a; Kale et al. 2020). In addition, the reservoir provides water for both agricultural activities and domestic use (Kale & Acarlı 2019a, 2019b).
Crayfish samples were collected monthly from the reservoir using fyke nets between July 2020 and June 2021. The samplings were conducted during the daytime. The fyke nets were left at the bottom of the reservoir and collected after 3 days. The monthly sample size was 30 individuals for both females and males. The collected specimens were secured in styrofoam boxes and transferred to the Biochemistry Lab of the Faculty of Marine Sciences and Technology at Çanakkale Onsekiz Mart University (Çanakkale, Türkiye) for the laboratory experiments and analyses. The shell and meat of female and male crayfish were extracted and homogenised separately according to sex.
The samples were extracted by methanol to find out the antioxidant radical scavenging capacity. Determining the free radical scavenging capacity requires using DPPH (2,2-Difenil-1-Pikrilhidrazil) as a stable and synthetic radical, and the antioxidant activity is determined by measuring the ability of the antioxidant to catch free radicals. DPPH is a dark purple coloured radical and its colour can be lightened after its reduction by antioxidant matter, which is the most widely used test method to measure the absorbance of the reaction of DPPH with the antioxidant at 515-517 nm (Brand-Williams et al. 1995; Huang et al. 2005). The analysis of DPPH entailed a mixture of a given amount of DPPH solution with the sample solution, reading the absorbance at 515 nm after 30 minutes and calculated according to the following formula (Equation 1):
IC50 is the value to represent 50% of the inhibition concentration of those concentrations shown by the DPPH radical scavenging effect.
To determine the total carotenoid content, we used the extraction method used by Yanar et al. (2004) and Zheng et al. (2010). Freeze-dried samples were exposed to sequential extraction with extracted samples being centrifuged to finally measure them by UV spectrophotometer. Spectrophotometric measurements were performed according to Oliveira et al. (2010) and Biehler et al. (2010) and Lichtenthaler & Buschmann (2001) by 450 and 470 nm, respectively. Three different calculation methods (Car1, Car2, Car3) were used to calculate carotenoid as given below (Equations 2-4) according to Oliveira et al. (2010), Biehler et al. (2010), and Lichtenthaler & Buschmann (2001), respectively.
The normal distribution of data was analysed using the Kolmogorov-Smirnov test of normality (
The radical scavenging effect was studied in meat samples of female and male crayfish with the DPPH method, and the relevant results are presented in Table 1. The IC50 value indicates the radical scavenging capacity. The better the radical scavenging effect, the lower the IC50 value. The IC50 value was the lowest in May with 122 mg g-1 in female individuals, while the highest value was in July with 388 mg g-1. On the other hand, male individuals showed the lowest and highest values of IC50 in July and November, respectively.
IC50 values (mg g-1) for the DPPH scavenging effect in the meat of female and male crayfish
Month | IC50 (mg g-1) | |
---|---|---|
Female | Male | |
July | 388.77 | 41.94 |
August | 291.18 | 62.49 |
September | 264.05 | 58.19 |
October | 334.00 | 157.33 |
November | 259.21 | 257.71 |
December | 231.95 | 134.89 |
January | 222.46 | 75.77 |
February | 236.40 | 56.45 |
March | 160.67 | 155.53 |
April | 232.14 | 63.16 |
May | 122.65 | 113.31 |
June | 134.51 | 85.77 |
Mean | 239.83 | 105.21 |
SD | 74.26 | 59.78 |
SD is the standard deviation
The study measured the total carotenoid content in the meat and shells of crayfish individuals. Table 2 illustrates the total carotenoid content in the samples collected over 12 months and calculated by different methods. The highest and the lowest values in female meat samples were found to be 21.26-3.38 μg g-1 (Car1) with a mean of 14.35 μg g-1. Consequently, it can be said that a mean value of 14.35 μg g-1 in the sample is a moderately good total carotenoid content in female individuals. Meat samples from the male individuals were calculated as 12.78 μg g-1 and it was concluded that there was no difference between female and male individuals. However, the carotenoid content in the shells of female and male individuals varied between 23 and 25 μg g-1. Comparison of the carotenoid of the shell with that of the meat indicates an almost two-fold difference between them – therefore, more carotenoids in the shell than in the meat samples.
Monthly variation of carotenoid content in the meat and shells of female and male crayfish
Month | Car1 (μg g-1) | Car2 (μg g-1) | Car3 (μg g-1) | |||
---|---|---|---|---|---|---|
Female | Male | Female | Male | Female | Male | |
Meat | ||||||
July | 6.76 ± 0.06 | 5.32 ± 0.40 | 6.84 ± 0.06 | 5.39 ± 0.40 | 7.29 ± 0.27 | 4.91 ± 0.53 |
August | 20.68 ± 0.85 | 22.78 ± 2.91 | 20.94 ± 0.86 | 23.06 ± 2.95 | 15.08 ± 1.55 | 21.51 ± 1.25 |
September | 20.62 ± 1.39 | 21.98 ± 1.78 | 20.88 ± 1.40 | 22.25 ± 1.80 | 25.13 ± 1.38 | 24.45 ± 1.56 |
October | 14.42 ± 0.48 | 11.28 ± 0.85 | 14.60 ± 0.49 | 11.42 ± 0.86 | 17.44 ± 0.76 | 11.32 ± 0.08 |
November | 21.26 ± 1.73 | 15.42 ± 1.22 | 21.53 ± 1.75 | 15.61 ± 1.23 | 26.76 ± 2.39 | 16.55 ± 1.18 |
December | 3.38 ± 0.03 | 4.84 ± 0.40 | 3.42 ± 0.03 | 4.90 ± 0.40 | 3.73 ± 0.10 | 3.22 ± 0.20 |
January | 10.04 ± 1.87 | 12.54 ± 1.22 | 10.17 ± 1.89 | 12.70 ± 1.23 | 10.01 ± 0.15 | 11.27 ± 1.62 |
February | 11.28 ± 0.06 | 9.98 ± 0.20 | 11.42 ± 0.06 | 10.10 ± 0.20 | 14.45 ± 2.59 | 8.64 ± 1.76 |
March | 13.20 ± 2.09 | 12.60 ± 2.04 | 13.36 ± 2.12 | 12.76 ± 2.06 | 13.71 ± 0.14 | 11.61 ± 0.48 |
April | 15.82 ± 1.10 | 13.50 ± 2.23 | 16.02 ± 1.12 | 13.67 ± 2.26 | 17.36 ± 0.34 | 14.26 ± 2.98 |
May | 18.10 ± 3.82 | 11.62 ± 2.12 | 18.33 ± 3.87 | 11.77 ± 2.15 | 17.45 ± 0.23 | 9.71 ± 1.00 |
June | 16.64 ± 1.13 | 11.46 ± 2.01 | 16.85 ± 1.15 | 11.60 ± 2.03 | 16.60 ± 1.12 | 7.53 ± 1.26 |
Mean | 14.35 | 12.78 | 14.53 | 12.94 | 15.42 | 12.08 |
SD | 5.69 | 5.43 | 5.76 | 5.50 | 6.55 | 6.30 |
Min | 3.38 | 4.84 | 3.42 | 4.90 | 3.73 | 3.22 |
Max | 21.26 | 22.78 | 21.53 | 23.06 | 26.76 | 24.45 |
Shell | ||||||
July | 23.30 ± 2.69 | 25.52 ± 2.15 | 23.59 ± 2.72 | 25.84 ± 2.18 | 26.53 ± 3.07 | 30.60 ± 3.38 |
August | 25.92 ± 3.45 | 31.44 ± 1.75 | 26.24 ± 3.49 | 31.83 ± 1.78 | 23.96 ± 1.91 | 37.40 ± 0.63 |
September | 31.14 ± 3.59 | 25.76 ± 2.04 | 31.53 ± 3.64 | 26.08 ± 2.06 | 36.11 ± 5.35 | 29.83 ± 2.72 |
October | 38.96 ± 1.81 | 17.94 ± 3.14 | 39.45 ± 1.83 | 18.16 ± 3.18 | 44.09 ± 2.17 | 18.15 ± 1.72 |
November | 37.98 ± 1.27 | 17.58 ± 3.54 | 38.45 ± 1.29 | 17.80 ± 3.58 | 43.40 ± 1.09 | 18.00 ± 2.35 |
December | 24.30 ± 0.25 | 24.52 ± 4.58 | 24.60 ± 0.26 | 24.83 ± 4.64 | 26.54 ± 1.51 | 26.63 ± 2.53 |
January | 18.32 ± 1.30 | 21.28 ± 0.85 | 18.55 ± 1.32 | 21.55 ± 0.86 | 20.17 ± 1.38 | 21.32 ± 3.21 |
February | 19.38 ± 1.90 | 18.80 ± 0.23 | 19.62 ± 1.92 | 19.03 ± 0.23 | 21.09 ± 1.73 | 20.69 ± 0.79 |
March | 18.70 ± 0.65 | 23.86 ± 0.31 | 18.93 ± 0.66 | 24.16 ± 0.32 | 20.94 ± 0.67 | 26.95 ± 1.41 |
April | 20.54 ± 1.33 | 27.72 ± 0.57 | 20.80 ± 1.35 | 28.07 ± 0.57 | 22.31 ± 0.71 | 32.61 ± 1.12 |
May | 24.08 ± 0.57 | 22.78 ± 2.69 | 24.38 ± 0.57 | 23.06 ± 2.72 | 26.57 ± 0.36 | 22.93 ± 3.35 |
June | 25.86 ± 3.37 | 25.98 ± 2.57 | 26.18 ± 3.41 | 26.30 ± 2.61 | 27.04 ± 6.57 | 29.72 ± 2.18 |
Mean | 25.71 | 23.60 | 26.03 | 23.89 | 28.23 | 26.24 |
SD | 6.98 | 4.16 | 7.07 | 4.22 | 8.40 | 6.10 |
Min | 18.32 | 17.58 | 18.55 | 17.80 | 20.17 | 18.00 |
Max | 38.96 | 31.44 | 39.45 | 31.83 | 44.09 | 37.40 |
SD is the standard deviation; Min is the minimum value; Max is the maximum value
The highest carotenoid content was observed in the meat of female and male individuals in November and September, and in the shells of female and male individuals in October and August, respectively (Table 2). While relatively higher carotenoid content values were observed in the meat in August and September compared to other months, it was found in shells in November and September.
Total carotenoid content in the shell and meat samples varied by month (
Pearson correlation between IC50 and radical DPPH calculated by different equations for the carotenoid content in the meat of female and male crayfish
♀ | IC50 | Car1 | Car2 | Car3 | ♂ | IC50 | Car1 | Car2 | Car3 |
---|---|---|---|---|---|---|---|---|---|
IC50 | 1 | IC50 | 1 | ||||||
Car1 | -0.206 | 1 | Car1 | 0.018 | 1 | ||||
Car2 | -0.206 | 1.000** | 1 | Car2 | 0.018 | 1.000** | 1 | ||
Car3 | -0.148 | 0.959** | 0.959** | 1 | Car3 | 0.005 | 0.971** | 0.971** | 1 |
Correlation is significant at the 0.01 level (2-tailed)
Pearson correlation between radical DPPH calculated by different equations for the carotenoid content in the shells of female and male crayfish
♀ | Car1 | Car2 | Car3 | ♂ | Car1 | Car2 | Car3 |
---|---|---|---|---|---|---|---|
Car1 | 1 | Car1 | 1 | ||||
Car2 | 1.000** | 1 | Car2 | 1.000** | 1 | ||
Car3 | 0.976** | 0.976** | 1 | Car3 | 0.984** | 0.984** | 1 |
Correlation is significant at the 0.01 level (2-tailed)
IC50 (mg g-1) is used to determine antioxidant capacity via/through the DPPH method (Kizilkaya et al. 2021). The antioxidant mechanism of carotenoids is often known as radical scavenging, a method based on measuring the ability of the antioxidants to scavenge radicals. Now that the process is simple, easy, fast and can be used even with a small-scale sample, it is an in-vitro method frequently preferred when considering antioxidant activity, which requires DPPH to be used only without adding any substrate, otherwise free radicals tend to attach directly to it (Fawwaz et al. 2020). It can be said that the IC50 found is inversely proportional to the antioxidant capacity (Manuguerra et al. 2020). The lowest values IC50 found were 122.65 mg g-1 and 41.94 mg g-1 in the meat of female and male crayfish individuals, respectively. The highest values of IC50 were seen in July as 388.77 mg g-1 and 257.71 mg g-1 for female and male individuals, respectively. This refers to the lowest radical scavenging effect. In other words, the highest antioxidant capacity was observed in female crayfish in March, April, May and June. Although antioxidant capacity varies continuously in male individuals, it can be said that it is higher in the summer months. During the study, it was determined that female individuals lost their granule-like appearance due to the increase in the size of the eggs in the body in July, August, September and October. Consumption or storage of antioxidants in the bodies of crustaceans is associated with whether they are in the reproduction period or hormonal activity, seasonal changes and the quality and quantity of food they eat (Valgas et al. 2020). It has been observed that female individuals tried to consume more energy to mature their eggs, particularly during the summer period of the study. Therefore, the IC50 value was high in this period whereas antioxidant capacity remained low due to increased energy consumption. Male individuals require less energy to produce sperm than females do for oogenesis, which could cause a lower IC50 value in males than females during the study. Marine carotenoids show strong antioxidant and reparative, antiproliferative and anti-inflammatory effects and can be used as nutraceutical/cosmeceutical agents for photoprotection of the skin from solar UV radiation or for the prevention of diseases caused by oxidative stress (Nichols & Katiyar 2010; Berthon et al. 2017). Diets rich in carotenoids have been attributed to the reduction in risks of some chronic and degenerative diseases such as cancer, cardiovascular disorders (Nishino 1998), cardiovascular disorders (Sesso et al. 2004) and age-related molecular degeneration (Zeegers et al. 2001). Carotenoids are a group of yellow-orange pigments divided into two general classes known as hydrocarbon carotenoids and xanthophylls in the form of oxidised derivatives. There are more than 650 carotenoids found naturally in bacteria, fungi, plants and animals (Matsuno 2001). Carotenoids from marine animals have different structures, most of which can be obtained from β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxantin and astaxanthin (Maoka 2011). Studies have shown that carotenoids are abundant in edible species of molluscs, crustaceans and echinoderms among marine organisms (Kantha 1989; Matsuno 2001; Maoka 2011; Wade et al. 2017; Kizilkaya et al. 2021). Carotenoids are strong antioxidants, so they have the capacity to scavenge free radicals (Miki 1991). Carotenoids are generally synthesised by photosynthetic algae and plants, fungi and bacteria while other organisms obtain the carotenoids they need directly from foods or carotenoid precursors in their diets by modifying them via metabolic reactions (de Carvalho & Caramujo 2017). Crustaceans do not synthesise carotenoids, but instead obtain them directly or partially from foods by modifying them through metabolic reactions (Castillo et al. 1982).
The total carotenoid content in freshwater crayfish varies according to the species and the organ in which it is stored (Czeczuga & Czeczuga-Smeniuk 1999), reproductive activity (Sagi et al. 1995), growth and, finally, nutrient abundance and diversity (D’Abramo et al. 1983). The present study revealed that carotenoid content in the meat and shells of female and male individuals varied according to months, this can be attributed to the variable consumption of carotenoid reserves during periods of active feeding, oogenesis in females and, finally, mature individuals shedding their skin. Seasonal differences in the total amount of carotenoids could be influenced by the quality and quantity of ingested food, as well as direct and indirect nutrition/feeding (Yanar et al. 2004). In the present study, the total carotenoid content was found to be 3.38 μg g-1 in females and 4.84 μg g-1 and males in meat samples, in the winter when the water temperature is low. Freshwater crayfish tend to abandon active feeding and retreat to their nests at temperatures below 10°C, when they do not ingest food from outside during this period. Carotenoids are stored in various organs (muscle, carapace, hepatopancreas, etc.) of the body (Sagi et al. 1995; Czeczuga & Czeczuga-Smeniuk 1999). The stored carotenoid is used when feeding activity is low to optimise health during developmental processes or in response to environmental stress such as pollution or parasites (Barim & Karatepe 2010; Caramujo et al. 2012; Wade et al. 2017). Czeczuga & Czeczuga-Semeniuk (1999) compared the total carotenoid contents in the meat and carapaces of
Free radicals are high-energy reactive molecules containing one or more unpaired electrons, formed as a result of natural body functions such as respiration and digestion. It is known that the free radicals formed are associated with cancer, atherosclerosis, Alzheimer, Parkinson, premature ageing and some chronic diseases. Antioxidants are capable of scavenging free radicals and preventing cell damage. In recent years, interest in aquatic products, which have an important place in terms of healthy nutrition, has been increasing and attracting great attention in healthy diets. Crustaceans have high levels of carotenoids in their waste as well as in their soft tissues.
Growth and reproduction activities in crayfish differ depending on seasonal changes (McLay & van den Brink 2016). The reproduction activity of
Marine invertebrates have long been investigated for their pharmacologic activities or other bioactive characteristics used in the biomedical field. As natural products of marine origin are becoming increasingly attractive as a result of current and potential processes in the pharmaceutical industry, the identification of new sources of those materials is of great importance (Pachaiyappan et al. 2014). It has been widely accepted that oxidative stress caused by an imbalance between prooxidants and antioxidants in an organism is mainly attributed to the root cause of chronic diseases (Urquiaga & Leighton 2000). Oxidative stress occurs due to the imbalance between the proliferation of Reactive Oxygen Species (ROS) and the antioxidant capacity of the organism. ROS could also occur during immune system reactions caused by external factors such as UV radiation. The proliferation of ROS occurs during normal psychological processes in a variety of cellular divisions as well (Zglińska et al. 2022). As a consequence of potential damage caused by this ROS reactivity to cells and tissues, marine organisms and other living things balance the proliferation of these radicals through various cellular antioxidant defensive mechanisms (Correia et al. 2003). Antioxidants consist of a large number of compounds and bioactive molecules based on a spectrum of sources (Reddy et al. 2011). Those obtained from natural sources play an important role in the neutralisation of oxidative stress (Sasikumar et al. 2009). Therefore, antioxidants have gained more importance and popularity due to their positive effects in the treatment of cardiovascular deficiencies, atherators, atherosclerosis, many types of cancer, ageing and related geriatric processes.
The main chemical components of crayfish shell waste are calcium carbonate, proteins, and chitin (Nguyen 2021). Among these components, chitin is considered to be the most valuable biomass material from crayfish shells (Shamshina et al. 2019). Efficient treatment and use of crayfish shell waste is critical for achieving sustainable development. Therefore, the extraction of chitin from crayfish shell waste is beneficial for solving the problem of crayfish shell waste accumulation, as well as further improving the value-added utilisation of crayfish shell waste (Li et al. 2023). Crayfish shells are a source of chitin, an antioxidant, and a potential source of beneficial dietary fiber (Bai et al. 2023). Chitin is a valuable biomaterial because of its antioxidant and antimicrobial properties, biocompatibility, non-toxicity, and biodegradability (Benhabiles et al. 2012). It is commonly used in food, water treatment, pharmacy medicine, and textiles (Felse & Panda 1999; Rinaudo 2006; Aranaz et al. 2009; Muzzarelli 2011; Kaya et al. 2015; Laribi-Habchi et al. 2015). Harish Prashanth & Tharanathan (2007) noted that chitin shows promise in a wide range of application, such as food and nutrition (food preservation and antioxidants), medical science (drugs and pharmaceuticals), microbiological (antibacterial), immunological (gene therapeutics and immune potentiator), and material science (packaging films and polymeric membranes). Chitosan is also broadly used in many sectors, mainly in food packaging, environmental protection, pharmaceutical, cosmetics, medicine, dentistry, textile, agriculture, veterinary, chemistry, and biotechnology (Kucukgulmez et al. 2016; Tekelioglu et al. 2017; Mirtajaddini et al. 2021; Farivar et al. 2022; Kadak et al. 2023). Moreover, carotenoids and carotenoproteins are responsible for most of the colour found in the exoskeletons and shells of crayfish and other crustaceans (Schuster 2020). Therefore, crayfish shells can be used to produce astaxanthin pigments used in the trout farming industry by colouring eggs and flesh. On the other hand, crayfish can be used as a biological indicator of exposure to both organic and inorganic pollution in aquatic systems (Schilderman et al. 1999). Crayfish are suitable biomarkers of heavy metal contamination of freshwater ecosystems due to their rapid bioaccumulation and long retention times (Kouba et al. 2010). Refaat Mohamed Morsi et al. (2023) used crayfish chitosan composite modified film, prepared from the exoskeleton of
In conclusion, this study investigated antioxidant radical scavenging capacity and total carotenoid content in the meat and shells of the narrow-clawed crayfish (