Antibacterial Activity in the Accessory Nidamental Gland Extracts of the Indian Squid, Loligo Duvauceli Orbigny

Indian Journal of Marine Sciences Vol. 39 (1), March 2010, pp. 100-104

Antibacterial activity in the accessory nidamental gland extracts of the Indian squid, Loligo duvauceli Orbigny

Gomathi, P. Nair, J.R* & Sherief, P.M. College of Fisheries, Kerala Agricultural University, Panangad, Kochi-682506, India. *[E-mail: [email protected]]

Received 18 November 2008; revised 9 April 2009

Antibacterial activity of accessory nidamental gland-butanol extracts from the Indian squid Loligo duvauceli in different stages of maturity was studied. The activity was evaluated by disc-diffusion method using five strains of bacteria. The extracts from ripe stage ANG showed antibacterial activity against gram negative bacterial strains, Escherichia coli and Pseudomonas aeruginosa , and gram positive bacteria, Staphylococcus aureus. Immature and spent gland extracts did not show any antibacterial activity. Spectrophotometric analysis of the ripe gland extract showed the maximum absorbance at 498.5 nm. This infers the presence of carotenoid pigments which impart the orange red colour to the ripe glands. Thin layer chromatography of the ANG-butanol extract revealed the presence of lipid components such as phospholipids, cholesterol, free fatty acids, triglycerides, fatty acid esters, and cholesteryl esters. The total free fatty acid content was significantly higher in the ripe ANG (16.0 ± 0.143 mg oleic acid/g tissue), in comparison to the immature ANG (10.3 ± 0.114 mg oleic acid/g tissue). Gas chromatographic studies of immature and ripe stages revealed the presence of a mixture of fatty acids. The major unsaturated fatty acids content in the ripe stage was 1.973 mg/gm tissue, whereas in immature stage it was only 0.251 mg/g tissue. Significantly higher levels of unsaturated fatty acids in the ripe stage could be the factor responsible for the antibacterial activity of the ANG-butanol extract.

(Key words: squid, accessory nidamental glands, antibacterial activity, chromatography, fatty acids)

Introduction The present study reports the antimicrobial activity Natural bioactive substances have the least quantum and characterization of the antimicrobial agent of of side effects when compared to synthetic products. ANG-butanol extracts from different maturity stages Although most antibiotics have been derived from of the Indian squid, Loligo duvauceli, caught from the terrestrial life, it is the marine world that may provide southwest coast of India. the pharmaceutical industry with the next generation of medicines. The biochemistries of seemingly simple Materials and Methods marine organisms such as blue green algae, sponges and Females of the Indian squid, Loligo duvauceli in squid are inspiring new ideas for drug development 1. different stages of maturity (immature, ripe and spent Accessory nidamental glands (ANG) are present in stages) were collected from the fish processing plant, the sexually mature females of cephalopods. These Bhatson’s Aquatic Products, Aroor, Alleppey District, paired glands are located at the anterior end of the Kerala, during the peak period of maturity nidamental glands and are closely associated with the (September-November, 2007). The animals were ventral surface of ink sac (Fig. 1). These glands are brought to the laboratory in ice cold condition. The reported to harbour dense bacterial community and the accessory nidamental glands were dissected out and ANG-bacterial community association has been removed aseptically. The color and weight of the studied in loliginids and sepioids 2,3,4,5,6,7,8,9 . Because of ANGs were noted. The glands were kept frozen ° its close association with the nidamental glands and the at -20 C till further use. Bacterial strains were oviduct, the ANG has long been thought to play a role obtained from National Institute of Oceanography, in protecting the eggs by coating them with symbiotic Regional Centre, Kochi. All the strains were bacteria or bacteriosins to ward of pathogens or maintained on nutrient agar. predators. The antibacterial properties of the ANG Exhaustive method of extraction with butanol 11 extracts from L. pealei 10 and from Sepia aculeata, was used with 2.5 g ANG in 5.0 ml butanol. The S. pharaonis and Sepiella inermis 11 have been reported. supernatant of the extract was filter sterilized. The NAIR et al . : ANTIBACTERIAL ACTIVITY IN THE ACCESSORY NIDAMENTAL GLAND 101

extract was stored at -20 °C for the antibacterial assay were used as the positive and negative controls and spectrophotometric, thin layer chromatographic respectively. (TLC) and gas liquid chromatographic (GLC) The absorbance spectra of the ANG-butanol analyses. extracts were measured on a spectrophotometer The antibacterial activity was tested by filter paper (Jasco, Japan) at 200–600 nm 2. disc diffusion technique 12 . Filter paper discs of 6 mm The butanol extract of the ANG in different size (Whatman No.1 filter paper) were impregnated maturity stages were further tested for polyphenols with 250 µl of ANG extract and the discs were /flavonoids by using 1% alcoholic ferric chloride as a vacuum dried. The activity was measured as the spray agent after TLC on silica gel G with butanol: diameter of the inhibition zone in mm. Standard acetic acid:water (4:1:5v/v/v) as a solvent system 13 . chloramphenicol discs and butanol impregnated discs Quinones were tested using 10% alcoholic potassium hydroxide as a spray agent. TLC was also carried out for the separation and identification of the lipid components in the ANG-butanol extract with hexane: ether:acetic acid (85:15:2 v/v/v) as the solvent system. The plates after drying were sprayed with 50% sulphuric acid and the separated lipid components were identified using standards 11 . The free fatty acid (FFA) content of the immature and ripe stage ANG extract was also determined 14 . For this, a known volume of extract was evaporated to dryness and re-dissolved in neutralized 95% ethanol. This was titrated against 0.01 N NaOH solution using phenolphthalein as an indicator. End point was formation of pink color that persisted for 30 seconds. The fatty acid composition of the butanol extract of immature and ripe ANG was analysed. The quantitative derivatisation of fatty acids to fatty acid methyl esters (FAME) for the ultimate analysis using GLC was carried out using boron triflouride- 15 methanol reagent (BF 3-CH 3OH) .

Results and Discussion Antimicrobial activity of immature, ripe and spent ANG-butanol extracts of Loligo duvauceli are given in Table 1. Antibacterial activity of ANG-butanol extract from different stages of maturity was tested against three gram negative and two gram positive Fig. 1—Loligo duvauceli- Viscera (IG-Ink gland; ANG- Accessory nidamental gland; NG-Nidamental gland; G-Gill; bacterial strains. In L. duvauceli , it was found that the O-Ovary) ANG-butanol extract from ripe animals showed Table 1—Antimicrobial activity of immature, ripe and spent ANG-butanol extracts of Loligo duvauceli Stages of maturity Escherichia coli Aeromonas hydrophila Pseudomonas Staphylococcus Bacillus megaterium aeruginosa aureus Immature Nil Nil Nil Nil Nil Ripe 7 mm Nil 7 mm 7 mm Nil Spent Nil Nil Nil Nil Nil Control* 30 mm 40 mm 12 mm 42 mm 32 mm Control** Nil Nil Nil Nil Nil

*Chloramphenicol-Positive control **Butanol-Negative control 102 INDIAN J. MAR. SCI., VOL. 39, NO. 1, MARCH 2010

antibacterial activity against Escherichia coli, whereas lipid components such as phospholipids, Staphylococcus aureus and Pseudomonas aeruginosa . cholesterol, free fatty acids, triglycerides, fatty acid The activity correlated well with the ripe stage of esters, and cholesteryl esters were detected (Fig. 2). females collected during the peak period of spawning. Similar findings were reported for the ripe stage ANG Extracts from the immature and spent stage glands did of S. pharaonis 11 . not show any antibacterial activity. The results of the free fatty acid estimation The ANG-butanol extract of L. pealei could inhibit indicated that FFA content was significantly higher the growth of marine pathogens like Vibrio (at 1% level, N=6) in ripe glands (16.0 ± 0.143 mg anguillarum and Streptomyces griseus 10 . Antibacterial oleic acid/g tissue) when compared to immature ones activity in the ANG-butanol extracts of Sepia (10.3 ± 0.114 mg oleic acid/g tissue). FFAs are aculeata , S. pharaonis and Sepiella inermis against known to have antimicrobial activity against various Escherichia coli , Aeromonas sp , Staphylococcus microorganisms 17,18,19,20,21 . In cuttlefish, S. pharaonis, aureus , and Bacillus megaterium have been FFA content of the ANGs in different maturity stages reported 11 . Among the cuttlefish species, extract from increased with advancement in maturity. Within the S. aculeata showed the highest activity and among the ripe glands, those with higher color intensity showed different organic solvents used, the butanol extract higher FFA content and correspondingly higher gave the highest activity. antibacterial activity than those with lower color The absorption spectra of the extracts from the intensity 22 . immature, ripe and spent accessory nidamental glands The results of the fatty acid composition of the are given in Table 2. Spectrophotometric analysis of immature and ripe stages based on GLC are given in ripe ANG-butanol extract showed an absorbance Table 3. The total fatty acid content was 3.925 mg/g maximum at 498.5 nm, followed by shoulders at tissue in ripe stage and 0.704 mg/g tissue in immature 528.0, 290.5 and 315.5 nm. The absorbance spectrum stage showing the high gross value in the ripe stage. of pigments extracted from the accessory nidamental glands of L. duvauceli corresponded with that reported for L. pealei 2 and for L. forbesi 5. The peak at 498.5 nm (2.597A), seen only in the ripe stage could be attributed to the presence of carotenoid pigments which impart the orange red colour to the ripe gland. In the adult female cuttlefish, Sepia officinalis an orange red pigment was found concentrated in the ANG. The new carotenoid pigment was termed ‘sepiaxanthine’ 16 . The red coloration of the ANG of sexually mature squid, L. pealei was attributed to the bacteria resident within it 2. Symbiotic bacteria (Alteromonas strain) in L. pealei were orange-red pigmented 6. The major peak obtained at 286 nm for the immature and spent stages indicate the presence of peptides/proteins. Fig. 2 —Thin layer chromatography of the ANG-butanol extracts (Lanes - IM-Immature stage; RG-Ripening stage; R1, R2, R3- Thin layer chromatography of the butanol extract Samples of ripe stage, Lipid components-PL-Phospholipids; CH- from the ANG of L. duvauceli did not show the Cholesterol; FFA-Free fatty acids; TG-Triglycerides; FE-Fatty presence of polyphenols/flavonoids or quinones, acid esters;CE-Cholesteryl esters)

Table 2—λ max for different peaks of absorbance of pigments from ANG extracts of Loligo duvauceli Maturity stages of ANG λmax Immature 285.0 (0.871A) 340.5 (0.122A) 358.0 (0.098A) 400.5 (0.064A) Ripe 290.5 (1.314A) 315.5 (0.985A) 498.5(2.597A) 528.0 (2.111 A) Spent 286.5 (0.862A) 346.5 (0.091A) 360.5 (0.087A) 400.5 (0.076)

Values within the brackets indicate the respective absorbance NAIR et al . : ANTIBACTERIAL ACTIVITY IN THE ACCESSORY NIDAMENTAL GLAND 103

[ 24 Table 3—Fatty acid composition of ANG-butanol extracts of Streptococcus mutans . The possible mechanism of Loligo duvauceli antibacterial action of unsaturated fatty acids might be mg/g ANG tissue an alteration of cell membrane properties or generation of free radicals 19,24,25,26 . Fatty acids Immature Ripe

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