Alternative exploration of hyaluronic acid from marine superstore

 

Kanchana S and Arumugam M*

Center of Advanced Study in Marine Biology, Faculty of Marine Science,

Annamalai University, Portnovo, Tamil Nadu, India

*Corresponding Author E-mail: mamnplab@gmail.com

 

ABSTRACT:

Hyaluronic acid (HA) is non-sulphated, linear glycosaminoglycans (GAGs) extensively used in biomedical, cosmetic and nutraceutical field. This type of GAGs has created a more attraction to biologists for exploration of HA from various sources. Consequently, this present study was focused towards the marine mollusk for alternative source target for the isolation of HA from the gastropod- Hemifusus cochlidium. The whole body tissues were defatted by acetone and pellet was extracted using digestion buffer followed by proteolytic treatment. Then the crude GAGs were subjected to anion exchange column for purification. Further, HA, D- glucuronic acid and N- acetyl glucosamine content was measured using the calorimetric method. Fourier transform infrared spectroscopy (FTIR) and Nuclear magnetic resonance (H1 NMR) were used for characterization. The yield from H. cochlidium and the presence of functional group in the isolated HA was characterized by (FTIR) and H1 NMR. In this investigation, the results suggested that marine gastropod could be an alternative source of HA.

 

KEY WORDS: Marine gastropod, Hyaluronic acid, FTIR, H1NMR, polysaccharide.

 

 

INTRODUCTION:

Marine environment is an exceptional reservoir of bioactive natural products, many of which exhibit structural or chemical features not scientifically evidence located within terrestrial natural products. It offers a tremendous biodiversity to discover useful therapeutic compounds that largely depend on the species specificity. The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans (GAGs), is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics [1]. GAGs have created a new arena of research owing to their several applications in the biomedical, veterinary, pharmaceutical and cosmetic field. GAGs include hyaluronic acid, heparin, heparansulphate, dermatan sulphate, chondroitin sulphate and kertansulphate. The structure and organization of glycosaminoglycans and proteoglycans from tissues of several vertebrates and invertebrates of marine organism have been extensively studied [2].

 

Hyaluronic acid (HA) is the most versatile compound also referred as “hyaluronan”. Hyaluronan, an anionic linear polysaccharide was formerly known as acid mucopolysaccharide and are now called as glycosaminoglycans [3]. It is a high molecular weight polysaccharide of the order of 10⁵ to 107KDa [4] with an un-branched backbone composed of alternating sequences of 𝛽 (1– 4) glucuronic acid and (1–3) –N-acetylglucosamine moieties and widely distributed in vertebrates as well as invertebrates. It is distinct from other GAGs by the devoid of sulfated groups and non - covalently linked peptide in their structure [5, 6]. HA is a building block for new biocompatible and eco-friendly polymers that have application in drug delivery, tissue engineering and viscosupplementation [7]. Concurrently, it also has been used in a medical, cosmetics and food industry [8]. So far HA was isolated from various sources include human umbilical cord, skin, rooster combs and bacteria [9]. In that human umbilical cord have the huge amount of HA and its cost expensive due to their source. This attempt of isolation of HA from marine source was carried out to provide with alternative sources and to change the source target for HA isolation.

 

MATERIALS AND METHOD:

Sample collection

The marine gastropod H. cochlidium were collected in the mudasaloodai landing center along the east coast of India. Samples were freshly collected and transferred to the laboratory in ice cold temperature and then the whole body tissues were dissected out from shells using the forceps.

 

Isolation of GAGs

The HA was isolated by the method of Volpi and Maccari [10]. The 250 gm of whole body tissue was dissected out and defatted with acetone for 24 h at 4ºC. The defatted content was centrifuged at 10,000g for 10 min; the pellets were dried at 60ºC for 24 h. Subsequently, 2.5 g of pellet was hydrated (1g/20ml) in digestion buffer containing 100mM sodium acetate at pH 5.5, 5mM EDTA, 5mM cysteine and papain 100mg/g of tissue and incubated for 24 h at 60ºC with continuous stirring. After boiling for 10 min, the mixture was centrifuged at 5000g for 15 min, and three volumes of ethanol saturated with sodium acetate was added to the supernatant and stored at 4ºC for 24 h. The precipitate was recovered by centrifugation at 5000g for 15 min and dried at 60ºC for 6 h.

 

The crude GAGs extracted from the tissue was dissolved in 10 ml of 0.05 M NaCl and centrifuged at 10,000 g for 10 min, the supernatant were then subjected to anion exchange column chromatography using DEAE cellulose column equilibrated with the same elution buffer. Glycosaminoglycans were eluted with a linear gradient of NaCl (0.05-1.2M) with a flow rate of 1ml/min and the fractions of 2ml were collected. Active fractions were identified by uronic acid estimation [11]. The respective active fractions were pooled with two volumes of ethanol and precipitated at 4ºC.  After centrifugation at 10,000g for 10 min, the pellet was dried at 60ºC.

 

Determination of analytical composition

The microtitre plate method was used for quantification of Hyaluronic acid and Uronic acid [11, 12]. A serial dilution of standard and sample of 50 µl (1mg/ml) was placed in a 96 well plate. Subsequently, 200µl of 25mM sodium tetraborate in sulphuric acid solution was added. Then the plate was heated at 100ºC for 10 min in an oven. After cooling at room temperature for 15 min, 50 µl of 0.125% carbazole in absolute ethanol was carefully added. After heating at 100°C for 10 min in an oven and cooling at room temperature for 15min, the plate was read in a micro plate reader at 550nm. The hexosamine was determined following the method of Wagner [13].

 

FTIR Analysis

Dry powder of HA was subjected to IR spectroscopy (Shimadzu) to determine the presences of the different amino, carboxyl and hydroxyl group. One part of the sample was mixed with ninety nine parts of dried KBr and then compressed to prepare a salt disc (3mm diameter). The absorption was read between 500 and 4000cm-1.

1H -NMR spectroscopy

1H NMR spectra were obtained using a Bruker Avance 400 nuclear magnetic spectrometer (Bruker DRX 500 Rheinstetten, Germany) operated at 400 MHz. The samples were pre- lyophilised three times with D2O and finally prepared by dissolving 5mg in 0.6 ml of D2O at high level of deuteration. All chemical shifts were given in parts per million (ppm).

 

RESULTS AND DISCUSSION:

The whole tissue was defatted using organic solvents, followed by proteolytic digestion the crude GAGs was isolated from marine mollusk species. The crude GAGs was subjected to the anion exchange column (cellulose) and eluted with the 0.05 - 1.2 M NaCl gradient. Further the collected fractions were determined the uronic acid level at 550nm. While evaluating the uronic acid concentration in fractions highest peak indicates the presences of hyaluronic acid. The yield of crude GAGs and HA in gastropod H.cochlidium (250 mg/g and 1.96 mg/g). Earlier, the net yield of HA from marine bivalve M. galoprovincialis was reported as 6.2 mg/gm [10] and A. pleuronectus (4.2 mg/g) [14] which is relatively higher than the present yield. Giji et al [15] reported that HA from the marine stingray liver was found to be 0.81 mg/g which indicates the lower yield when compared to marine gastropod. Likewise, the yield of HA in horny layer of guinea pig was around 0.25 mg/gm [16]. In the present study showed that the yield of  H.cochlidium HA was higher than terrestrial source. But there exists the difference in the net yield of which may be attributed to species variation and extraction methods applied.

 

The analytical compositional of the HA were quantified through standard methods. The disaccharides, including uronic acid and N-acetyl glucosamine contents were estimated as 47.3 %, 32.1% of H. cochlidium respectively. Likewise, HA isolated from marine stingray liver and marine bivalve A. pleuronectus [14] also showed same range of uronic acid and N-acetyl glucosamine contents [15]. Further Dicker and Franklin [17] have revealed the HA disaccharides composition for cortex of fresh kidney from various sources such as pigs (42.7, 39.2), sheep (36.8, 35.4) and dogs (40.8, 37.3) of similar ranges respectively.

IR spectroscopy has been used successfully to characterize glycosaminoglycans. The functional group of HA from molluscan sample of H. cochlidium was studied in FT-IR spectroscopy and compared to HA from Human umbilical cord were shown in fig. 1. The Standard HA shows 10 major peaks between the ranges 500 and 4000cm-1.The sharp peak was observed in the region of 3442.94 cm, representing to the hydrogen bonded O-H stretching and N-H stretching vibration of the N-acetyl side chain. The peaks at 1633.71 and 1323.17 cm-1assigned to amide I group of C=O carboxyl and aromatic primary amine CN stretching respectively. The peak at 2924.09 cm-1 was obtained owing to the methyl C-H stretch responsible for glucuronic acid. The peak at 1029.99 cm-1 was observed for the primary alcohol C-O stretch. Alkyne C-H bonds were responsible


for 667.37 cm-1 stretch. Further, Alkradet al. [18] reported similar range of bands in the IR for HA digested with HAase which greatly supports our results. The structural difference between HA and Chondrotin sulphate is detected by the presence of –O-SO3- side groups absorbance at 1240 cm-1 [19]; therefore, the absorbance responsible for the –O-SO3 group was not ascertained. The FTIR results confirmed that the major functional peaks in the spectra are present in H. cochlidium as similar to standard HA.

 

Fig.1:  FTIR spectrum of isolated HA from H. cochlidium

 

Fig.2:  FTIR spectrum of standard HA

 

Proton NMR spectra of most organic compounds were characterized by chemical shifts in the range +14 to -4 ppm and by spin-spin coupling between protons. The peak responsible for the presence of glycosidic linkages at 4.5 ppm and N-acetyl glucosamine at 2 ppm was recorded for H. cochlidium (Fig.3). The α/β anomeric protons of the reducing end of N-acetyl glucosamine peaks were observed at chemical shifts between 5.2-5.3 ppm. Our results significantly agree with with the earlier reports of chemical shifts observed for HA isolated from a marine vertebrate source [15]. Further, the presence of reducing ends and glycosidic linkages in acid hydrolyzed HA [20] considerably concurs with HA from mollusk samples. The presences of methyl groups were identified in the shielded region between 0.999 and 1.769 which concur with the earlier reports on proton NMR spectra of HA isolated from bacterial sources [21].

 

 

Fig.3: H1NMRspectrum of isolated HA from H. cochlidium

 

 

CONCLUSION:

Recently the stipulate for HA in industrial scale has grown remarkably. Therefore, the search for source target for isolation of HA has been focused in marine superstore. Owing to this we have isolated HA from cheaper sources of marine gastropods and characterized its structure using NMR and IR spectroscopy. These results concluded that marine mollusk could serve as an alternative source for HA.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Department of Biotechnology (DBT) (G4/8855) for providing financial support and to the authorities of Annamalai University for providing facilities.

 

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Received on 12.10.2014          Accepted on 09.11.2014        

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Asian J. Pharm. Res. 4(4): Oct.-Dec.2014; Page 169-173