Medicinal and therapeutic properties of cephalopod ink : a short review

Cephalopoda is under the phylum of Mollusca with several important marine oceanic habitats. They are mostly well-known for their different uses such as food ingredients and nutraceutical benefits. Among all the different habitats of this class, different species of squid and cuttlefish often containing various nutraceutical properties along with the use as a food item. Surprisingly, the ink of these different species of cephalopods also contains different nutraceutical properties which are thrown away as by-product by most of the processing industries and consumers. There are different studies which focused on these properties obtained from the ink of these cephalopods. These studies clearly showed that this ink is a great source for decreasing various health problems and can be used widely in both pharmaceuticals and food industries. The aim of this review is to focus on its potentials and make it an evident towards the future uses of cephalopod ink.


Introduction
Cephalopods live in the marine environment and the number of cephalopod species is relatively low (approximately 700) compared to the number of other species; however, they are widely distributed in different marine habitats (Nair and Sherief, 2010;Derby, 2014).Nautiloidea (nautilus) and Coleoidea are the two major groups that represent cephalopods (Derby 2014), which include squids, cuttlefish, octopus and nautilus (Nair and Sherief, 2010).Among those, squid is the major constituent of the Cephalopoda class (Normal et al., 2002).Currently, squid and cuttlefish are an important fishery product all over the world, especially in Southeast Asian countries (Hoque et al., 2010).Cephalopods have historically been used as human food, especially among Greeks and Egyptians, in different ways (Sundaram, 2009).
Primarily in India, cephalopods were used in the dried form (Rao, 1954;Vijayakumaran, 1984).In the Philippines, cephalopods are first boiled in vinegar and are then fried in oil and spices (Voss, 1963).Squids are incorporated with fish stew in Britain to increase taste (Cornell and Handy, 1982).The Japanese have mastered the use of different varieties of cephalopods in various dishes (Silas et al., 1985).Moreover, other varieties of cephalopods are popular as food dishes food such as pickles (Sundaram, 2009).
During the processing of squids and cuttlefish, the viscera and ink sac containing the ink are considered byproducts and are potential threats for creating serious ecological problems as well as environmental pollution without proper management.These by-products can be a potential source of bioactive compounds and have been proven to be an alternative medicine with a wide range of therapeutic applications.The utilization of these byproducts will benefit the processing industry as well as reduce serious ecological problems and pollution (McConnell et al., 1993;Karim et al., 2016).
Most cephalopods, excluding nautiloid, have ink sacs and thus produce ink (Hanlon and Messenger, 1996).Cephalopod species that live in low light, including deep sea areas, produce and use ink (Bush et al., 2007).Even when cephalopods are small and young, ink sacs can be present and produce ink (Boletzky, 1987).In Japan, squid ink has traditional applications in food products (Nishimoto et al., 1980).In addition, it is believed that 'Ika-shiokara,' which is a cured cuttlefish meat produced in Japan exhibits anti-septic effect when cuttlefish ink added to it (Takai et al., 1993).Cephalopod ink has already been reported for its various therapeutic values (Takai et al., 1992).The crude ink extracts from various species of cephalopods have been studied for their antimicrobial, preservative, antioxidant, anti-cancer, antiretroviral and many other properties (Mochizuki, 1979;Takai et al., 1992;Takaya et al., 1994;Sasaki et al., 1997;Rajaganapathi et al., 2000;Russo et al., 2003;Sadok et al., 2004;Yang et al., 2005;Lei et al., 2007;Girija et al., 2008).These properties make cephalopod ink attractive.In this review, we attempt to combine all these properties to determine the potential prospects of using cephalopod ink in different ways.

Cephalopods ink and its components
Cephalopod ink is a natural substance discharged by cephalopods from their ink sac when they confront enemies and try to escape from predators (Ortonne et al., 1981;Lei et al., 2007).The release of dark ink is used as a defensive means to avoid enemies and risks (Liu et al., 2011;Nicomrat and Tharajak, 2015).The ejected ink helps cephalopods to confuse predators and sends a signal to other cephalopods about the danger (Lucero et al.,1994).
Cephalopod ink consists of a suspension of melanin granules in a viscous colourless medium.In the mantle cavity, the ink gland cells of the digestive tract degenerate and shed their content into the ink sac, which is used as a reservoir for the ink.The production and ejection of the ink seem to be regulated by the glutamate/ nitric oxide/cGMP signaling pathway located in the ink gland (Palumbo et al., 1997;Palumbo et al., 2000;Liu et al., 2011).In addition to a large amount of melanin, the ink also contains proteins, lipids, glycosaminoglycans and various metals (Copper, Cadmium).(Lei et al., 2007;Liu et al., 2011;Zhong et al., 2009).It also contains a variety of melanogenic enzymes, including tyrosine, which is a dopachrome-rearranging enzyme (Palumbo et al., 1998).

Antimicrobial activity
Different studies have been conducted to determine the antimicrobial activities of different squid inks.Nirmale et al. (2002), suggested that the freeze-dried and precipitated ink of the Indian squid Loligo duvauceli has good antibacterial effects.It mostly showed strong antibacterial effects against gram-negative bacteria, Salmonella spp.Escherichia coli, Vibrio cholerae, V. parahaemolyticus and Pseudomonas spp.However, the effects of the gram-positive bacteria Staphylococcus spp.and Micrococcus spp.are weaker than the effects against gram-negative bacteria.Giriji et al. (2011), reported on a novel antimicrobial protein, Lolduvin-s, which was isolated from the ink of Indian squid (Loligo duvauceli) and showed promising antibacterial and antifungal activities against different pathogens.Girija et al. (2014), revealed that squid (L.duvauceli) ink extract has antibacterial potential against dental caries pathogens.It has also been reported that squid ink has good antibacterial properties against extended spectrum betalactamase (ESBL)-producing strains of E. coli and Klebsiella pneumonia (Girija et al., 2012).Karim et al. (2016) suggested that squids treated with 0.25% squid ink showed very low growth of bacteria during storage at 4°C.Nicomrat and Tharajak (2015) found that squid (L.duvauceli) and soft cuttlefish (Sepioteuthis lessoniana) ink have strong antimicrobial activity against biofilms causing microorganisms.Mochizouki (1979), reported that cuttlefish ink has an inhibitory effect on Staphylococcus aureus and has antiseptic properties.The ink of pharaoh cuttlefish, Sepia pharaonic, has antibacterial effects against human pathogens such as Pseudomonas aeruginosa, Staphylococcus epidermidis, K. pneumonia and E. coli.In these cases, researchers have found that crude ink extracted in hexane and column-purified ink extracted in diethyl ether show maximum inhibitory effects against these pathogens (Nithya et al., 2011).Vennila et al. (2010), reported that cuttlefish (Sepia aculeate) ink and squid (L.duvauceli) ink have antifungal effects against Fusarium spp.and Aspergillus fumigates.Diaz and Thilaga (2016), revealed that crude and partially purified ink extracts of squid (L.duvauceli) and cuttlefish (Sepia pharaonis) have antibacterial effects against eight different bacterial strains.Table 1 represents a brief information about different in research on antimicrobial properties of cephalopod ink.Ink from different species of cephalopods had been used for studying antimicrobial properties of the ink.Various pathogenic bacteria and microorganisms are used in these studies, a list of that microorganisms also included in Table 1.In most of the studies, different kinds of ink samples showed prominent antimicrobial activities against most of the pathogenic bacteria which made cephalopod ink a very good antimicrobial agent and thus it became an object of attraction among researchers.

Anti-cancer activity
Squid and cuttlefish ink has the potential to act as anticancer agents, and this is based on in vitro studies of cancer cell line.The anti-cancer effects of squid and cuttlefish ink occur through the initiation of apoptosis and are affiliated with different chemicals of ink (Derby, 2014).

REVIEW
• 100 µL crude ink concentration of both squid and cuttlefish had good activity against all sample microorganism.• 20 µL concentration had good activity only against V. fischeri (squid ink) and K. pneumonia, A. hydrophila (cuttlefish ink).• Only two fractions (30% and 40%) of partially purified squid ink and one fraction (80%) partially purified cuttlefish ink exhibited activity.
• Hexane extract of squid ink showed good antibacterial activity against both organisms.Diaz et al. (2014), evaluated the anti-cancer activity of squid (L.duvauceli) ink.Crude and partially purified squid ink was used on the Hep G2 cell line, and cell viability and cell proliferation assays were performed.They revealed having good anti-carcinogenic activity of partially purified L. duvauceli ink on the HepG2 cell line.Takaya et al. (1994), reported on the antitumour peptidoglycan fraction of squid ink obtained from Illex argentinus.Tris-HCl buffer (pH 6.8) was used for the extraction and the fractionation of the ink to obtain the peptidoglycan fraction.Strong antitumour activity was found by applying it against Meth-A fibrosarcoma in BALB/c mice.As the fraction of squid ink has no direct cytotoxic effect against Meth-A cells, the stimulation of host-mediated responses may be the reason for the suppression of tumour growth.Sasaki et al. (1997), also studied the antitumour activity of the peptidoglycan fraction of squid (I.argentinus) ink, which was delipidated in acetone and used against Meth-A tumours from BALB/c mice, and they found 64% cure rate against Meth-A tumours.Later, Naraoka et al. (2000) showed that there are two different components that are responsible for the anti-tumour activity of squid ink.They suggested that illexin-peptidoglycan, tyrosinase and the complex of these two are responsible for the antitumour activity of squid ink, and the complexity of the two components showed the highest antitumour activity against Meth-A tumour cells of BALB/c mice.Zhong et al. (2009) worked on the protective effects of squid ink in chemotherapy.In the study, BALB/c mice were used as animal models, and injuries were induced by cyclophosphamide.This study showed positive results for the protection of the haemopoietic system from chemotherapeutic injury and suggested that it could be employed to develop cell protective drugs for use in clinical treatment of tumours.Chen et al. (2010) reported that chemically sulphated polysaccharides (SIPs) isolated from squid (Ommastrephes bartrami) ink have good antitumour activity against HepG2 tumour cells.Both in vitro and in vivo studies provide substantial proof that SIPs have potential compounds for the prevention of tumour metastasis.Senan et al. (2013a) suggested that ink extracts of different cuttlefish species, such as Sepia pharaonis, Sepia aculeate, Sepiella inermis and squid (L.duvauceli), that were delipidated in acetone and extracted by Tris-HCl buffer have potential antiproliferative effects against chick embryo fibroblast cells, which provides proof of the potential use of the ink extracts as an anticancer agent.Another study by Senan et al. (2013b) provided information about the anticancer activity of the purified C2 fraction of S. pharaonis ink against cervical cancer cell lines-HeLa and Caski.Fahmy and Soliman (2013) reported that cuttlefish (Sepia officinalis) ink extract has cytotoxic activity and can be used as promising anticancer drugs, which was

Mode of ink preparation
Microorganism Results References Squid (L.duvauceli) • Aqueous extract of crude ink of squid, cuttlefish, and octopus were used.
• Fusarium spp.(mold) • Aspergillus fumigates (mold) • Squid, cuttlefish and octopus ink showed good antifungal activity.REVIEW determined using the sulphorhodamine B (SRB) method against hepatocellular carcinoma (HepG2) cell line.Soliman et al. (2015) found that S. officinalis ink extract had anticancer effects against Ehrlich ascites carcinoma (EAC) in Swiss albino mice.He added that Sepia ink extract inhibits tumour growth in the ascites tumour model.Russo et al. (2003) applied melanin-free S. officinalis ink on various cell lines, including PC12 cells, and found that it was cytotoxic to the PC12 cell lines and could be used in future carcinogenic drugs.
Guo-Fang et al. (2011) performed enzymatic hydrolysis of S. officinalis ink using trypsin enzyme and isolated an oligopeptide from hydrolysates, which have anticancer activities.These potential anticancer properties were proved when the isolated oligopeptide from Sepia ink hydrolysates were used against human prostate carcinoma cells (DU-145).Huang et al. (2012) reported that Sepia ink oligopeptide (SIO), which is a tripeptide extracted from Sepia esculenta ink, inhibits prostate cancer inducing SIO was applied against three human prostate cell lines DU-145, PC-3 and LNCaP to prove the potency of SIO as an anticarcinogenic agent.Wang et al. (2008) applied sulphated Sepiella maindroni ink polysaccharide (SIP-SII) to human ovarian carcinoma cells SKOV3 and human umbilical vein vascular endothelial cells ECV304, and their results suggested that SIP-SII might oppress the migration and invasion of carcinoma cells via inhibition of matrix metalloproteinases-2 (MMP-2) proteolytic activity.Zong et al. (2013) found that SIP-SII has anti-metastatic and anti-angiogenic properties, which also result in the depression of the invasion and migration of carcinoma cells.Changlong et al. (1999) revealed that squid ink and its extracts might activate immunity of cells by stimulating natural killer cells and macrophages to kill tumour cells indirectly.Table 2 represents the different studies on anticancer properties of cephalopods ink.Most of the anticancer studies of cephalopods ink were done against different cell lines to measure the anticancer properties.The ink had been used in a different mode and good anticarcinogenic effect was found in almost all cases shown in Table 2.

Antioxidant activity
Squid and cuttlefish ink have antioxidant properties that reside in both the melanin and melanin-free fractions of the ink (Derby, 2014).Liu et al. (2011) studied the antioxidant activity of squid ink on growing broiler chickens by mixing ink in their diet.Total SOD (Superoxide dismutase) activity and MDA (malondialdehyde) content determination results showed strong antioxidant abilities of squid ink.Vate and Benjakul (2013) studied the melanin-free ink from the splendid squid Loligo formosana.They performed differently in vitro antioxidant tests, including DPPH radical scavenging activity, ABTS radical scavenging activity, ferric reducing antioxidant power (FRAP) and chelating activity towards Fe 2+ , and found good antioxidant value in the melanin-free squid ink.Sun et al. (2011) studied the polysaccharides extracted from squid ink by alkaline protease and tested for the antioxidant activities using DPPH, radical scavenging and a FRAP assay and found a high amount of antioxidant value from the extracted polysaccharides.Chen et al. (2007) removed the melanin from squid ink, and a free radical scavenging activity test was conducted on the removed melanin.They showed that squid melanin scavenge hydroxyl free radical remarkably which are indicators of the antioxidant properties of squid melanin.Lin and Chen (2005) also isolated melanin from cuttlefish (Sepia) ink and investigated the antioxidant properties, and they found high antioxidant value.Lei et al. (2008) also found antioxidant effects of melanin-Fe squid ink when they were using it as a treatment for iron deficiency anaemia (IDA) in rats.Fahmy and Soliman (2013) investigated the antioxidant activity of cuttlefish (S. officinalis) ink extract using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging and lipid peroxidation assays and measured promising antioxidant properties from the Sepia ink extract.Another study by Soliman et al. (2015) was conducted to compare the antioxidant and anti-cancer activities to 5-fluorouracil (5-Fu) in vivo, where Swiss albino mice were used as experimental animals, and they found good antioxidant properties.
An in vivo study was conducted by Saleh et al. (2015) and provided information on the complications of hepatic fibrosis associated with bile duct ligation (BDL) and the potential curative role of S. officinalis ink extract in hepatic damage induced by BDL.The result showed a significant reduction in oxidative stress, which proves the antioxidant activity of the ink extracts.Another in vivo study provided information that cuttlefish ink increases the antioxidant level in mice (Lei et al., 2007).Studies on antioxidant properties of cephalopod inks are listed in Table 3.Both in vitro and in vivo studies had been done to determine the antioxidant properties of cephalopod ink.The mode of antioxidant studies is given in Table 3.

Other properties of cephalopod ink
Squid (L.duvauceli) and cuttlefish (Sepiella inermis) ink have antiretroviral activities, which were reported by Rajaganpathi et al. (2000).Squid ink has anti- • Gel filtrated melanin free ink sulfated by PySO 3 • MTT assay • The human hepatocellular liver carcinoma cell line (HepG2) • Showed dose-dependent suppression of cell invasion and migration in HepG2.• Can be a potential candidate for the prevention of tumor metastasis.
Chen et al. ( Cuttlefish (S. pharaonis) • Crude ink delipidated with acetone then gel filtrated to get purified peptidoglycan fraction • MTT assay   Mimura et al. (1987).Low molecular weight melano proteins were obtained from Ommastrephes bartrami ink and studied for anti-inflammatory effects using carrageenan-induced rat paw oedema.In addition, Fahmy and Soliman (2013) also found anti-inflammatory effects of cuttlefish (Sepia) ink.Kim et al. (2003) extracted and purified an angiotensin-converting enzyme from squid ink which can dilate blood vessels and lower blood pressure.According to Mimura et al. (1982) squid ink has strong anti-ulcerogenic properties.Melanin extracts constitute almost 90% of squid ink and could inhibit gastric secretion in rats.The molecular weight of melano protein that is contained in the active fractions might be responsible for the anti-ulcerogenic activity, by enhancing the glycoprotein activity of gastric mucosa.In addition to the above properties, Sepia ink also has antineoplastic properties (Soliman et al., 2015) and plasma coagulation properties (Vennila et al., 2010) which make it an attractive element among researchers.

Conclusion
Compared to synthetic products, natural bioactive products have the fewest side effects, and the marine environment can be a good source of natural bioactive products.Among different marine products, cephalopod ink is one of the best sources of bioactive products.Different studies which were demonstrated in this study provide information about the neutraceuticals properties of the cephalopods ink.This review also collaborates information about various modes of using the ink with methodology, which can be helpful for future researchers to conduct a new research.Functional and nutraceutical properties of cephalopods ink are mainly focused in this study so that awareness on using cephalopods ink can be built up.In the near future, it is hoped that this can reduce the wastage of the ink industrially.Proper consciousness on its variety of medicinal and therapeutic properties will make the ink an attractive object for preparing functional food and alternative medicine.

Table 1 .
• Antimicrobial properties of cephalopods ink REVIEW

Table 3 (
Continued).Antioxidant properties of cephalopod ink REVIEW