- Email: [email protected]
Chitin and chitosan preparation from shrimp shells Penaeus monodon and its human ovarian cancer cell line, PA-1
Accepted Manuscript Title: Chitin and Chitosan preparation from shrimp shells Penaeus monodon and its human Ovarian Cancer Cell Line, PA-1 Authors: Haripriya Srinivasan, Kanayairam Velayutham, Ramanibai Ravichandran PII: DOI: Reference:
S0141-8130(17)32659-4 http://dx.doi.org/10.1016/j.ijbiomac.2017.09.035 BIOMAC 8212
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
21-7-2017 29-8-2017 13-9-2017
Please cite this article as: Haripriya Srinivasan, Kanayairam Velayutham, Ramanibai Ravichandran, Chitin and Chitosan preparation from shrimp shells Penaeus monodon and its human Ovarian Cancer Cell Line, PA-1, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitin and Chitosan preparation from shrimp shells Penaeus monodon and its human Ovarian Cancer Cell Line, PA-1 Haripriya Srinivasan, Kanayairam Velayutham and Ramanibai Ravichandran* Unit of Aquatic Biodiversity, Department of Zoology, University of Madras, Guindy Campus, Chennai- 600 025, Tamil Nadu, India *
Corresponding author: [email protected]; [email protected]
Tel.: 91-44-22202833; Fax.: 044-22300899 Abstract In the present study, chitin and chitosan preparation from shrimp shells Penaeus mondon and its ovarian cancer cell line (PA-1). FTIR spectrum sharp absorption peak at 1655 cm−1 is assigned to ketone C=O (α) unsaturated with chitosan. X-ray diffraction showed the presence of chitin and chitosan were strongest peak at 18.91° (β) and 29.75º (α) characters. SEM observations of chitin and chitosan surface morphologies of P. monodon showed that microfibril and porous structures. Anticancer activity of chitin and chitosan against human ovarian cancer cell line showed that chitosan an exhibited notable higher activity than chitin. Anticancer activity of aquacultural waste of shrimp shells mediated chitosan, which was proved to be good novel pharmaceutical industries. Key words: Chitin; Chitosan; Penaeus monodon; FTIR Introduction Chitin and its derivatives are biomolecules of a great potential, possessing versatile biological activities, demonstrating biocompatibility and biodegradability
[1]
. Chitin production
includes krill, crayfish, insects, clams, oysters, jellyfish, algae and fungi. Few researchers worked on the chitin content of shrimp Parapenaeus longirostris [5]
. Benhabiles et al.
[6]
[2-4]
and Bacillus licheniformis
reported that chitin possesses antibacterial properties. Further, have 1
evaluated the recovery of protein during the process of obtaining chitin from shrimp shells
[2]
and oligomers and monomers are obtained from it [7]. Chitosan was natural, non-toxic, biodegradable, copolymer of glucosamine and N-acetylglucosamine prepared from chitin by deacetylation. It is found commercially in the waste products of the marine food processing industry
[8-9]
. Chitosan were tested few biological
application antimicrobial [10] and hypocholesterolemic [11]. Cancer is the most important public health burden in both developed and developing countries. Approximately 8, 50, 000 new cancer cases are being diagnosed and about 5, 80, 000 cancer related death occurs every year in India [12]. Death due to cancer worldwide is projected to rise with an estimated 12 million deaths in 2030. Around 25 million persons are living with cancer around the world [13]. Ovarian cancer is one of the most predominant cancers found in women particularly post menopause stage. Ovarian cancer ranks fifth in cancer deaths among women, accounting for more deaths than any other cancer of the female reproductive system. Her lifetime chance of dying from ovarian cancer is about 1 in 100 (Cancer facts sheet, 2017). Ovarian cancer can be of three origins which includes epithelial, stromal and germ line origin. Worldwide 22,440 women are affected with ovarian cancer per year and 14,080 deaths are reported which gives an alarming percentage of about 62 %. In Chennai, around 480 cases were reported for ovarian cancer of malignant nature [14]. Globally, the waste generated during the industrial processing of shrimp is about 40–50 % of its total weight depending on the species and consists of heads, shells and tails
[15]
.
Production of shrimp in India accounts 1,00,000 tons per year [16] and 35-45 % of this amount are waste (Head/thorax). Only 5 % of shrimp waste is actually used in some way, mostly for animal 2
feed, the remainder is discarded and represents a major environmental problem
[17]
. In the
present study, chitin and chitosan preparation from shrimp shells Penaeus monodon and its human ovarian cancer cell line, PA-1. 2. Materials and methods 2.1. Sample collection Penaeus monodon is widely consumed as sea food in various parts of Chennai city and one of the prominent shrimps sold in the fish markets of the city. P. monodon shrimp shells were collected from Light house and Chepauk fish markets. P. monodon organisms were collected were identified with the aid of an eminent taxonomist. 2.2. Sample preparation The shrimp shells were washed thoroughly under running tap water followed by washing with distilled water to remove all the intact proteins, tissues, soluble organics and inorganics and other impurities. The shells were then collected and boiled in water for 1 hour to remove the adherent tissues. The shells are then oven dried at 160 ºC in hot air oven for 2 hours so as to make them brittle which in turn helps to break the crystalline structure of chitin. Post 2 hours the shells were then ground to fine powder using a standard mixer-grinder [6]. 2.3. Demineralization The main inorganic compound of the shell includes calcium carbonate and various other trace elements. In order to remove calcium carbonate only dilute hydrochloric acid is used to prevent chitin hydrolysis
[18]
. 1M of hydrochloric acid was added to shell powder and constantly
stirred at 150 rpm for 75 minutes at room temperature. The ratio of dried shells to acid solution used for demineralization is 1/30 (w/v). The demineralized shells were then filtered and collected
3
in a whatmann filter paper No. 1, washed to neutrality with distilled water thrice and oven dried at 80ºC for 24 hours [6]. 2.4. Deproteinization Shrimp shell powder was then subjected to the same process similar to demineralization for deproteinization. Adherent proteins were removed with sodium hydroxide. 3M of sodium hydroxide was added to demineralized shell powder and constantly stirred at 150 rpm for 75 minutes at room temperature. The ratio of demineralized shells to sodium hydroxide solution for deproteinization is 1/30(w/v). The demineralized shells were then filtered and collected in whatmann filter paper, washed to neutrality with distilled water thrice and dried for 24hours in the oven at 80 ºC to get the chitin residue [6]. 2.5. Decolourisation The chitin residue is decolourized with acetone and sodium hypochlorite. Chitin residue is first treated with acetone in solid to solvent ratio of 1/10(w/v), stirred for ten minutes at 150 rpm at room temperature. Then it is filtered with whatmann filter paper, dried at room temperature for 2 hours. Chitin residue is then treated with 0.3155 sodium hypochlorite for 15 minutes at 150 rpm at room temperature with solid to solvent ratio of 1/10(w/v). The chitin is now decolourised which is filtered, washed with distilled water and dried in the hot air oven at 40 ºC for 16 hours. The chitin is then stored in air tight containers [6]. 2.6. Deacetylation Chitosan Kurita
[19]
is
converted
from
chitin
by
deacetylation
process
suggested
by
. 1 g of chitin is treated with 50 ml of 50 % sodium hydroxide. The mixture was
constantly stirred at a fixed temperature of 90 ºC. After 50 minutes post the occurrence of a foamy texture the mixture was filtered, neutralized with distilled water. The chitosan residue is 4
then treated with 80% Alcohol with solid to solution ratio of 1/30(w/v) followed by oven drying at 80ºC for 24 hours. The chitosan is then stored in air tight containers [20]. 2.7 Characterization of chitin and chitosan Dry powder of chitin and chitosan were used for FTIR analysis. These measurements were carried out on a Perkin-Elmer Spectrum One instrument in the diffuse reflectance mode at a resolution of 4 cm−1in KBr pellets. XRD (Bruker AXS D8) is one of the most powerful and established technique for material structural analysis capable of providing information about the structure of a material at the atomic level. SEM analysis based upon a beam of high energy electrons to generate a variety of signals at the surface of solid specimens. The extracted chitin and Chitosan was analysed by using JEOL, Model JFC-1600 scanning electron microscope. 2.8. Antioxidant activity The potential free radical scavenging activity of chitin and chitosan (1mg/ml) using DPPH (2,2-diphenyl-1-picrylhydrazyl) was estimated with slight modifications of Blois
[21]
was
assessed spectrophotometrically. 2.9. Chitin and chitosan against ovarian cancer cell line The cytotoxic effect of chitin and chitosan was tested against cancer cell line PA-1 by MTT assay
[22]
. The PA-1 ovarian cancer cell line was seeded in 96-well micro plates (1 x 106
cells/well) and incubated at 37°C for 24 hours in 5% CO2 incubator and allowed to grow to 90% confluence. Then the medium was replaced and the cells were treated with chitin and chitosan at various concentration of such as 5-50 µg/mL and incubated for 24 hours. For anticancer activity, a stock solution of chitin 1mg/ml in 20% ortho phosphoric acid and chitosan 1mg/ml in 10% ortho phosphoric acid was prepared The cells were then washed with phosphate-buffer saline (PBS, pH- 7.4) and 20 μL of (MTT) solution (5 mg/mL) was added to each well. The plates were 5
then allowed to stand at 37ºC in the dark for additional 4 hours. The formazan crystals were dissolved in 100 μL DMSO and the absorbance was read spectrometrically at 570 nm. The concentration that inhibited 50% of cell growth was referred as IC50 value, which was used as a parameter for cytotoxicity study. The morphological changes of untreated (control) and the cells treated at IC50 were observed under bright field microscope after 24 hours and photographed. 3. Result and discussion The yield of chitin and chitosan from P. monodon shrimp shell waste was 30% and 35%, respectively. Chitin was prepared by using acid and alkaline treatments; the yield of chitin was 30% in the total weight of the dried P. monodon shells, after deacetylation, the yield of chitosans were in the range of 35%. The cuttlebone of Sanguisorba officinalis was found to be 20% of chitin
[23]
, whereas in general, the cuttlefish reported 3% to 20% of chitin
[24]
. One of the major
struggle related to the preparation of pure chitins is keeping a structure as close as potential than the native form is to minimize the partial deacetylation and chain degradation caused by demineralization and deproteinization applied during process of the raw materials. 3.1. Fourier Transform Infrared Spectroscopy Infrared spectroscopy of the structure changes of initial chitin and chitosan were confirmed by FTIR spectroscopy (Fig. 1). FTIR spectra of chitin exhibited prominent peaks at 3447, 2918, 1655, 1379, 1073 cm−1 (black colour) and chitosan revealed that the peaks at 3428, 2921, 1636, 1420, 1096 cm−1 (red colour). The peak at 3447 cm−1 overlaps with N-H stretching whose functional group is amine with a medium intensity. The significant decrease of transmittance in this band region indicates that the C–H alkane vibration was affected by the attachment of chitin at 2918 cm−1strong intensity. The sharp absorption peak at 1655 cm−1 is assigned to ketone C=O, α and β unsaturated. The presence of the peak at 1379 cm−1 is the alkane 6
bending vibration of C- H groups with variable intensity and the peak at 1073 cm−1 confirms the presence of C–O stretching of alcohol. Salah et al.
[3]
investigated the deacetylation of chitin to
produce chitosan was recognized by increasing of NH2 functional groups (708 cm−1, 1572 cm−1 and 3111 cm−1) and by decreasing of C= O functional groups (1661.7 cm−1) for shrimp shells of P. longirostris. Kaya et al [25] have explained that chitin and chitosan with low crystalline values are more useful for wastewater treatment. Many other studies of chitin polymorphs have revealed differences in crystalline peaks between α, β and γ chitins obtained from various sources [26]. The suggests that chitin extracted Amide I band is split at 1667 cm−1, which indicates that this chitin from P. monodon shrimp shells was in α character. 3.2. X- Ray Diffraction X-ray diffraction pattern of chitin and chitosan extracted from P. monodon. The XRD exhibited intense peaks in the whole spectrum of 2θ value ranging from 20° to 80˚. Chitin was assigned to diffraction from the 18.91º, 25.73º and 38.47º. Jang et al.
[27]
reported that β-chitin
have crystalline peaks at 20.3°. In the XRD results of the present study, sharp peaks were observed at 18.91° had β character at chitin (Fig 2A). The peaks for chitosan were assigned to diffraction from the 29.75º, 34.85º, 37.62º, 40.93º, 46.03º and 47.88º. The strongest peak was at 37.62º and 29.75º had α character for chitosan (Fig 2B). Cárdenas et al
[28]
reported that XRD
patterns of α-chitin (chitins from shrimp, lobster, prawn and king crab) and β-chitin (chitin from squid) exhibited their major characteristic peak at 19.3° and 18.8° respectively. Yen and Mau [29] found that fungal chitin (γ-chitin) showed two crystalline reflections at 5.6° and 19.6°. Irrespective of their origin, the three types of chitin consistently display a major peak at ~19° in their crystalline structure.
7
3.3. Scanning Electron Microscope SEM images of chitin and chitosan revealed their surface, dense and firm morphology which helps us in a great deal to understand their mechanism of biological activities. The images of chitin (Fig. 3) and chitosan (Fig. 4) were taken between 65- 6000 magnification. The SEM images of chitin and chitosan exhibited microfibril and porous structures. Crab and shrimp chitin [30]
, Metapenaeus stebbingi (shrimp) chitosan
structures show porous structures.
[31]
and silkworm chrysalis chitin and chitosan
[32]
In the present study, both chitin and chitosan surface
morphologies of P. monodon showed that microfibril and porous structures. 3.4. Antioxidant activity Free radical-scavenging is a primary mechanism by which antioxidants inhibit oxidative processes. Both chitin and chitosan exhibited high antioxidant activity compared to ascorbic acid against DPPH and the scavenging activity (Fig. 5). The scavenging activity with various concentrations was ranging from 6.25 mg/ml to 1000 mg/ml varied from 5.41 % to 59.02 % in chitin and 11.35 % to 68.25 % in chitosan, respectively. Matsugo et al.
[33]
demonstrated the
moderate antioxidant properties of water-soluble chitosan derivatives in the inhibition of a radical chain reaction and they recommended water soluble acetylated chitosan as a new source of antioxidants. The results suggest that chitin and chitosan could be applied as proton donors and could react with free radicals to exchange them to more stable stuff and terminate the radical chain effect. 3.5. Anticancer activity The cytotoxic effects of chitin and chitosan on a human ovarian cancer cell line, PA-1 have been evaluated. The results indicate that chitin (Fig. 6) and chitosan (Fig. 7) exhibited cytotoxic effects at various concentrations of 10 to 50 µg/ml. The results, presented in Fig. 6, 8
indicate that chitin have the potential to suppress 100% of the growth of PA-1 tumour cells at concentration at 50 µg/ml. However, the chitosan has the potential to suppress 100% of the growth of PA-1 tumour cells at low concentration at 10µg/ml (Fig. 7). These results suggest that chitin and chitosan are able to develop an inhibitory effect on the proliferation of the PA-1 cell line. Bouhenna et al.
[4]
reported that chitin derivatives against human cancer cell lines RD and
Hep2. In the chitin was cytotoxic at IC50= 400 μg/ml against Hep2 cells lines. It concluded that the cytotoxicity is probably due to interactions among the negative charges groups of tumor cells and the positively charged groups of the chitin and chitosan. The same conclusion, as the previous authors, was given by Bouhenna et al.[4] who studied the cytotoxicity of chitin derivatives towards human cancer cell lines. In conclusion, the extracted chitin and chitosan were characterized by using FTIR, XRD and SEM analysis and the results revealed that the chitosan extracted from the exoskeleton of P. monodon is in alpha form. The findings of this study provide useful information for further studies to expand the biological applications of chitosan. Therefore, the results of this study that chitosan samples from P. monodon have significant anticancer properties against human ovarian cancer cell line, PA-1. Acknowledgements The authors are grateful thanks to the University of Madras, Department of Zoology for their help support in carrying out the present study. Reference 1. I. Younes, S. Hajji, V. Frachet, M. Rinaudo, K. Jellouli, M. Nasri, Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan, Inter. J. Biol. Macromol. 69 (2014) 489-98. 9
2. M.S. Benhabiles, N. Abdi, N. Drouiche, H. Lounici, A. Pauss, M.F.A. Goosen, N. Mameri, Protein recovery by ultrafiltration during isolation of chitin from shrimp shells Parapenaeus longirostris, Food Hydrocoll. 32 (2013) 28-34. 3. R. Salah, P. Michaud, F. Mati, Z. Harrat, H. Lounici, N. Abdi, N. Drouiche, N. Mameri, Anticancer activity of chemically prepared shrimp low molecular weight chitin evaluation with the human monocyte leukaemia cell line, THP-1. Inter. J. Biol. Macromol. 52 (2013) 333-339. 4. M. Bouhenna, R. Salah, R. Bakour, N. Drouiche, N. Abdi, H. Grib, H. Lounici1, N. Mameri, Effects of chitin and its derivatives on human cancer cells lines, Environ. Sci. Poll. Res. 22 (2015) 15579–15586. 5. H. Laribi-Habchi, A. Bouanane-Darenfed, N. Drouiche, A. Pauss, N. Mameri, Purification, characterization and molecular cloning of an extracellular chitinase from Bacillus licheniformis stain LHH100 isolated from wastewater samples in Algeria. Inter. J. Biol. Macromol. 72 (2015) 1117-28. 6. M.S. Benhabiles, R. Salah, H. Lounici, N. Drouiche, M.F.A. Goosen, N. Mameri, Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste. Food hydrocoll. 29 (2012) 48-56. 7. M. Dziril, H. Grib, H. Laribi-Habchi, N. Drouiche, N. Abdi, H. Lounici, A. Pauss, N. Mameri, Chitin oligomers and monomers production by coupling γ radiation and enzymatic hydrolysis, J. Ind. Eng. Chem. 26 (2015) 396-401. 8. Z. Limam, S. Selmi, S. Sadok, A. El Abed, Extraction and characterization of chitin and chitosan from crustacean by-products: Biological and physicochemical properties. African J. Biotechnol. 10(4) (2011) 640-647. 9. A. Khanafari, R. Marandi, S.H. Sanatei, Recovery of chitin and chitosan from shrimp waste by chemical and microbial methods. J. Environ. Health Sci. Eng. 5(1) (2008) 1-24. 10. W. Xia, P. Liu, J. Zhang, J. Chen, Biological activities of chitosan and chitooligosaccharides. Food Hydrocoll. 25 (2011) 170–79.
10
11. R. Jayakumar, D. Menon, K. Manzoor, S.V. Nair, H. Tamura, Biomedical applications of chitin and chitosan nanomaterials - a short review. Carbohydr. Polym. 82 (2010) 227-232. 12. M. Dhanamani, S.L. Devi, S. Kannan, Ethnomedicinal plants for cancer therapy–a review. Hygeia J.D. Med. 3(1) (2011) 1-10. 13. C. Foster, D. Fenlon, Recovery and self-management support following primary cancer treatment. British J. Cancer 105 (2011) 21-28. 14. V. Shanta, R. Swaminathan, Population based cancer registry, Chennai cancer institute (WIA), Adyar, Chennai. Individual Registry write up, (2006- 2008) pp: 165-183. 15. M. Ogawa, E.L. Maia, A.C. Fernandes, M.L. Nunes, M.I. Oliveira, S.T. Freitas, Waste from the processing of farmed shrimp: a source of carotenoid pigments. Ciência e Tecnologia de Alimentos, 27 (2) (2007) 333-337 16. K. Gopakumar, Textbook of fish processing technology. Indian Council of Agricultural Research, New Delhi (2002) pp: 467-483. 17. L.A. Cira, S. Huerta, G.M. Hall, K. Shirai, Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery. Proc. Biochem. 37 (2002) 1359-1366. 18. H. K. No , S. P. Meyers, Preparation and characterization of chitin and chitosan: a review. J. Aqua. Food Pro. Tech. 4 (1995) 27-52. 19. K. Kurita, Chitin and chitosan functional biopolymers from marine crustaceans, Mar. Biotechnol. 8(3) (2003) 203–226. 20. M.S. Benhabiles, N. Abdi, N. Drouiche, H. Lounici, A. Pauss, M.F.A. Goosend, N. Mameri, Fish protein hydrolysate production from sardine solid waste by crude pepsin enzymatic hydrolysis un a bioreactor coupled to an ultrafiltration unit, Mat. Sci. and Eng. C 32 (2012) 922-928. 21. M.S. Blois, Antioxidant determinations by the use of a stable free radical. Nature 181 (1958) 1199-1200. 22. T. Mossman, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Method. 65 (1983) 55-64. 11
23. L. Sun, K. Feng, R. Jiang, J. Chen, Y. Zhao, R. Ma, H. Tong, Watersoluble polysaccharide from Bupleurum chinense DC: Isolation, structural features and antioxidant activity, Carbohydr. Polym. 79 (2010) 180-183. 24. A. Tolaimate, J. Debrieres, M. Rhazi, A. Alagui, Contribution to the preparation of chitin and chitosan with controlled physicochemical properties, Polymer 44 (2003) 7939-7952. 25. M. Kaya, T. Baran, A. Mentes, M. Asaroglu, G. Sezen, K.O. Tozak, Extraction and characterization of alpha-chitin and chitosan from six different aquatic invertebrates, Food Biophys. 9 (2014) 145-157. 26. M.T. Yen, J.H. Yang, J.L. Mau, Antioxidant properties of fungal chitosan from shiitake stipes. LWT-Food Sci. Technol. 40 (2007) 225-261. 27. M.K. Jang, B.G. Kong, Y.I. Jeong, C.H. Lee, J.W. Nah, Physicochemical characterization of α‐chitin, β‐chitin and γ‐chitin separated from natural resources, J. Polym. Sci. Part A: Polym. Chem. 42 (2004) 3423–3432. 28. G. Cárdenas, G. Cabrera, E. Taboada, S.P. Miranda, Chitin characterization by SEM, FTIR, XRD, and 13C cross polarization/mass angle spinning NMR. J. Appl. Polym. Sci. 93 (2004) 1876-1885. 29. M.T. Yen, J.L. Mau, Selected physical properties of chitin prepared from shiitake stipes. LWT-Food Sci. Technol. 40 (2007) 558–563. 30. M.T. Isa, A.O. Ameh, J.O. Gabriel, E. Leonardo, Extraction and characterization of chitin from Nigerian sources, J. Pract. Technol. 21 (2012) 73-81. 31. A. Kucukgulmez, M. Celik, Y. Yanar, D. Sen, H. Polat, A.E. Kadak, Physicochemical characterization of chitosan extracted from Metapenaeus stebbingi shells, Food Chem. 126(3) (2011) 1144-1148. 32. A.T. Paulino, J.I. Simionato, J.C. Garcia, J. Nozaki, Characterization of chitosan and chitin produced from silkworm chrysalides, Carbohydr. Polymer. 64 (2006) 98–103. 33. S. Matsugo, M. Mizuie, M. Matsugo, R. Ohwa, H. Kitano, T. Konishi, Synthesis and antioxidant activity of water-soluble chitosan derivatives. Biochem. Mol. Biol. Inter. 44(5) (1998) 939-948. 12
Fig. 1 FT-IR analysis of chitin/ chitosan from Penaeus monodon
13
Fig. 2 XRD analysis (A) Chitin and (B) Chitosan from Penaeus monodon
14
Fig. 3 SEM analysis for chitin (A) 500 µm and (B- C) 10 μm
Fig. 4 SEM analysis for chitosan (A) 40 µm, (B) 20 µm and (C) 50 μm 15
Fig. 5 Radical scavenging activity of chitin/ chitosan for Penaeus monodon
16
Fig. 6 MTT assay of chitin extraction from P.monodon against PA-1 ovarian cancer cell line (A) 10 µg/ml, (B) 20 µg/ml, (C) 30 µg/ml, (D) 40 µg/ml and (E) 50 µg/ml.
17
Fig. 7 MTT assay of chitosan extraction from P.monodon against PA-1 ovarian cancer cell line (A)10 µg/ml, (B) 20 µg/ml, (C) 30 µg/ml, (D) 40 µg/ml and (E) 50 µg/ml.
18
S0141-8130(17)32659-4 http://dx.doi.org/10.1016/j.ijbiomac.2017.09.035 BIOMAC 8212
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
21-7-2017 29-8-2017 13-9-2017
Please cite this article as: Haripriya Srinivasan, Kanayairam Velayutham, Ramanibai Ravichandran, Chitin and Chitosan preparation from shrimp shells Penaeus monodon and its human Ovarian Cancer Cell Line, PA-1, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.09.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitin and Chitosan preparation from shrimp shells Penaeus monodon and its human Ovarian Cancer Cell Line, PA-1 Haripriya Srinivasan, Kanayairam Velayutham and Ramanibai Ravichandran* Unit of Aquatic Biodiversity, Department of Zoology, University of Madras, Guindy Campus, Chennai- 600 025, Tamil Nadu, India *
Corresponding author: [email protected]; [email protected]
Tel.: 91-44-22202833; Fax.: 044-22300899 Abstract In the present study, chitin and chitosan preparation from shrimp shells Penaeus mondon and its ovarian cancer cell line (PA-1). FTIR spectrum sharp absorption peak at 1655 cm−1 is assigned to ketone C=O (α) unsaturated with chitosan. X-ray diffraction showed the presence of chitin and chitosan were strongest peak at 18.91° (β) and 29.75º (α) characters. SEM observations of chitin and chitosan surface morphologies of P. monodon showed that microfibril and porous structures. Anticancer activity of chitin and chitosan against human ovarian cancer cell line showed that chitosan an exhibited notable higher activity than chitin. Anticancer activity of aquacultural waste of shrimp shells mediated chitosan, which was proved to be good novel pharmaceutical industries. Key words: Chitin; Chitosan; Penaeus monodon; FTIR Introduction Chitin and its derivatives are biomolecules of a great potential, possessing versatile biological activities, demonstrating biocompatibility and biodegradability
[1]
. Chitin production
includes krill, crayfish, insects, clams, oysters, jellyfish, algae and fungi. Few researchers worked on the chitin content of shrimp Parapenaeus longirostris [5]
. Benhabiles et al.
[6]
[2-4]
and Bacillus licheniformis
reported that chitin possesses antibacterial properties. Further, have 1
evaluated the recovery of protein during the process of obtaining chitin from shrimp shells
[2]
and oligomers and monomers are obtained from it [7]. Chitosan was natural, non-toxic, biodegradable, copolymer of glucosamine and N-acetylglucosamine prepared from chitin by deacetylation. It is found commercially in the waste products of the marine food processing industry
[8-9]
. Chitosan were tested few biological
application antimicrobial [10] and hypocholesterolemic [11]. Cancer is the most important public health burden in both developed and developing countries. Approximately 8, 50, 000 new cancer cases are being diagnosed and about 5, 80, 000 cancer related death occurs every year in India [12]. Death due to cancer worldwide is projected to rise with an estimated 12 million deaths in 2030. Around 25 million persons are living with cancer around the world [13]. Ovarian cancer is one of the most predominant cancers found in women particularly post menopause stage. Ovarian cancer ranks fifth in cancer deaths among women, accounting for more deaths than any other cancer of the female reproductive system. Her lifetime chance of dying from ovarian cancer is about 1 in 100 (Cancer facts sheet, 2017). Ovarian cancer can be of three origins which includes epithelial, stromal and germ line origin. Worldwide 22,440 women are affected with ovarian cancer per year and 14,080 deaths are reported which gives an alarming percentage of about 62 %. In Chennai, around 480 cases were reported for ovarian cancer of malignant nature [14]. Globally, the waste generated during the industrial processing of shrimp is about 40–50 % of its total weight depending on the species and consists of heads, shells and tails
[15]
.
Production of shrimp in India accounts 1,00,000 tons per year [16] and 35-45 % of this amount are waste (Head/thorax). Only 5 % of shrimp waste is actually used in some way, mostly for animal 2
feed, the remainder is discarded and represents a major environmental problem
[17]
. In the
present study, chitin and chitosan preparation from shrimp shells Penaeus monodon and its human ovarian cancer cell line, PA-1. 2. Materials and methods 2.1. Sample collection Penaeus monodon is widely consumed as sea food in various parts of Chennai city and one of the prominent shrimps sold in the fish markets of the city. P. monodon shrimp shells were collected from Light house and Chepauk fish markets. P. monodon organisms were collected were identified with the aid of an eminent taxonomist. 2.2. Sample preparation The shrimp shells were washed thoroughly under running tap water followed by washing with distilled water to remove all the intact proteins, tissues, soluble organics and inorganics and other impurities. The shells were then collected and boiled in water for 1 hour to remove the adherent tissues. The shells are then oven dried at 160 ºC in hot air oven for 2 hours so as to make them brittle which in turn helps to break the crystalline structure of chitin. Post 2 hours the shells were then ground to fine powder using a standard mixer-grinder [6]. 2.3. Demineralization The main inorganic compound of the shell includes calcium carbonate and various other trace elements. In order to remove calcium carbonate only dilute hydrochloric acid is used to prevent chitin hydrolysis
[18]
. 1M of hydrochloric acid was added to shell powder and constantly
stirred at 150 rpm for 75 minutes at room temperature. The ratio of dried shells to acid solution used for demineralization is 1/30 (w/v). The demineralized shells were then filtered and collected
3
in a whatmann filter paper No. 1, washed to neutrality with distilled water thrice and oven dried at 80ºC for 24 hours [6]. 2.4. Deproteinization Shrimp shell powder was then subjected to the same process similar to demineralization for deproteinization. Adherent proteins were removed with sodium hydroxide. 3M of sodium hydroxide was added to demineralized shell powder and constantly stirred at 150 rpm for 75 minutes at room temperature. The ratio of demineralized shells to sodium hydroxide solution for deproteinization is 1/30(w/v). The demineralized shells were then filtered and collected in whatmann filter paper, washed to neutrality with distilled water thrice and dried for 24hours in the oven at 80 ºC to get the chitin residue [6]. 2.5. Decolourisation The chitin residue is decolourized with acetone and sodium hypochlorite. Chitin residue is first treated with acetone in solid to solvent ratio of 1/10(w/v), stirred for ten minutes at 150 rpm at room temperature. Then it is filtered with whatmann filter paper, dried at room temperature for 2 hours. Chitin residue is then treated with 0.3155 sodium hypochlorite for 15 minutes at 150 rpm at room temperature with solid to solvent ratio of 1/10(w/v). The chitin is now decolourised which is filtered, washed with distilled water and dried in the hot air oven at 40 ºC for 16 hours. The chitin is then stored in air tight containers [6]. 2.6. Deacetylation Chitosan Kurita
[19]
is
converted
from
chitin
by
deacetylation
process
suggested
by
. 1 g of chitin is treated with 50 ml of 50 % sodium hydroxide. The mixture was
constantly stirred at a fixed temperature of 90 ºC. After 50 minutes post the occurrence of a foamy texture the mixture was filtered, neutralized with distilled water. The chitosan residue is 4
then treated with 80% Alcohol with solid to solution ratio of 1/30(w/v) followed by oven drying at 80ºC for 24 hours. The chitosan is then stored in air tight containers [20]. 2.7 Characterization of chitin and chitosan Dry powder of chitin and chitosan were used for FTIR analysis. These measurements were carried out on a Perkin-Elmer Spectrum One instrument in the diffuse reflectance mode at a resolution of 4 cm−1in KBr pellets. XRD (Bruker AXS D8) is one of the most powerful and established technique for material structural analysis capable of providing information about the structure of a material at the atomic level. SEM analysis based upon a beam of high energy electrons to generate a variety of signals at the surface of solid specimens. The extracted chitin and Chitosan was analysed by using JEOL, Model JFC-1600 scanning electron microscope. 2.8. Antioxidant activity The potential free radical scavenging activity of chitin and chitosan (1mg/ml) using DPPH (2,2-diphenyl-1-picrylhydrazyl) was estimated with slight modifications of Blois
[21]
was
assessed spectrophotometrically. 2.9. Chitin and chitosan against ovarian cancer cell line The cytotoxic effect of chitin and chitosan was tested against cancer cell line PA-1 by MTT assay
[22]
. The PA-1 ovarian cancer cell line was seeded in 96-well micro plates (1 x 106
cells/well) and incubated at 37°C for 24 hours in 5% CO2 incubator and allowed to grow to 90% confluence. Then the medium was replaced and the cells were treated with chitin and chitosan at various concentration of such as 5-50 µg/mL and incubated for 24 hours. For anticancer activity, a stock solution of chitin 1mg/ml in 20% ortho phosphoric acid and chitosan 1mg/ml in 10% ortho phosphoric acid was prepared The cells were then washed with phosphate-buffer saline (PBS, pH- 7.4) and 20 μL of (MTT) solution (5 mg/mL) was added to each well. The plates were 5
then allowed to stand at 37ºC in the dark for additional 4 hours. The formazan crystals were dissolved in 100 μL DMSO and the absorbance was read spectrometrically at 570 nm. The concentration that inhibited 50% of cell growth was referred as IC50 value, which was used as a parameter for cytotoxicity study. The morphological changes of untreated (control) and the cells treated at IC50 were observed under bright field microscope after 24 hours and photographed. 3. Result and discussion The yield of chitin and chitosan from P. monodon shrimp shell waste was 30% and 35%, respectively. Chitin was prepared by using acid and alkaline treatments; the yield of chitin was 30% in the total weight of the dried P. monodon shells, after deacetylation, the yield of chitosans were in the range of 35%. The cuttlebone of Sanguisorba officinalis was found to be 20% of chitin
[23]
, whereas in general, the cuttlefish reported 3% to 20% of chitin
[24]
. One of the major
struggle related to the preparation of pure chitins is keeping a structure as close as potential than the native form is to minimize the partial deacetylation and chain degradation caused by demineralization and deproteinization applied during process of the raw materials. 3.1. Fourier Transform Infrared Spectroscopy Infrared spectroscopy of the structure changes of initial chitin and chitosan were confirmed by FTIR spectroscopy (Fig. 1). FTIR spectra of chitin exhibited prominent peaks at 3447, 2918, 1655, 1379, 1073 cm−1 (black colour) and chitosan revealed that the peaks at 3428, 2921, 1636, 1420, 1096 cm−1 (red colour). The peak at 3447 cm−1 overlaps with N-H stretching whose functional group is amine with a medium intensity. The significant decrease of transmittance in this band region indicates that the C–H alkane vibration was affected by the attachment of chitin at 2918 cm−1strong intensity. The sharp absorption peak at 1655 cm−1 is assigned to ketone C=O, α and β unsaturated. The presence of the peak at 1379 cm−1 is the alkane 6
bending vibration of C- H groups with variable intensity and the peak at 1073 cm−1 confirms the presence of C–O stretching of alcohol. Salah et al.
[3]
investigated the deacetylation of chitin to
produce chitosan was recognized by increasing of NH2 functional groups (708 cm−1, 1572 cm−1 and 3111 cm−1) and by decreasing of C= O functional groups (1661.7 cm−1) for shrimp shells of P. longirostris. Kaya et al [25] have explained that chitin and chitosan with low crystalline values are more useful for wastewater treatment. Many other studies of chitin polymorphs have revealed differences in crystalline peaks between α, β and γ chitins obtained from various sources [26]. The suggests that chitin extracted Amide I band is split at 1667 cm−1, which indicates that this chitin from P. monodon shrimp shells was in α character. 3.2. X- Ray Diffraction X-ray diffraction pattern of chitin and chitosan extracted from P. monodon. The XRD exhibited intense peaks in the whole spectrum of 2θ value ranging from 20° to 80˚. Chitin was assigned to diffraction from the 18.91º, 25.73º and 38.47º. Jang et al.
[27]
reported that β-chitin
have crystalline peaks at 20.3°. In the XRD results of the present study, sharp peaks were observed at 18.91° had β character at chitin (Fig 2A). The peaks for chitosan were assigned to diffraction from the 29.75º, 34.85º, 37.62º, 40.93º, 46.03º and 47.88º. The strongest peak was at 37.62º and 29.75º had α character for chitosan (Fig 2B). Cárdenas et al
[28]
reported that XRD
patterns of α-chitin (chitins from shrimp, lobster, prawn and king crab) and β-chitin (chitin from squid) exhibited their major characteristic peak at 19.3° and 18.8° respectively. Yen and Mau [29] found that fungal chitin (γ-chitin) showed two crystalline reflections at 5.6° and 19.6°. Irrespective of their origin, the three types of chitin consistently display a major peak at ~19° in their crystalline structure.
7
3.3. Scanning Electron Microscope SEM images of chitin and chitosan revealed their surface, dense and firm morphology which helps us in a great deal to understand their mechanism of biological activities. The images of chitin (Fig. 3) and chitosan (Fig. 4) were taken between 65- 6000 magnification. The SEM images of chitin and chitosan exhibited microfibril and porous structures. Crab and shrimp chitin [30]
, Metapenaeus stebbingi (shrimp) chitosan
structures show porous structures.
[31]
and silkworm chrysalis chitin and chitosan
[32]
In the present study, both chitin and chitosan surface
morphologies of P. monodon showed that microfibril and porous structures. 3.4. Antioxidant activity Free radical-scavenging is a primary mechanism by which antioxidants inhibit oxidative processes. Both chitin and chitosan exhibited high antioxidant activity compared to ascorbic acid against DPPH and the scavenging activity (Fig. 5). The scavenging activity with various concentrations was ranging from 6.25 mg/ml to 1000 mg/ml varied from 5.41 % to 59.02 % in chitin and 11.35 % to 68.25 % in chitosan, respectively. Matsugo et al.
[33]
demonstrated the
moderate antioxidant properties of water-soluble chitosan derivatives in the inhibition of a radical chain reaction and they recommended water soluble acetylated chitosan as a new source of antioxidants. The results suggest that chitin and chitosan could be applied as proton donors and could react with free radicals to exchange them to more stable stuff and terminate the radical chain effect. 3.5. Anticancer activity The cytotoxic effects of chitin and chitosan on a human ovarian cancer cell line, PA-1 have been evaluated. The results indicate that chitin (Fig. 6) and chitosan (Fig. 7) exhibited cytotoxic effects at various concentrations of 10 to 50 µg/ml. The results, presented in Fig. 6, 8
indicate that chitin have the potential to suppress 100% of the growth of PA-1 tumour cells at concentration at 50 µg/ml. However, the chitosan has the potential to suppress 100% of the growth of PA-1 tumour cells at low concentration at 10µg/ml (Fig. 7). These results suggest that chitin and chitosan are able to develop an inhibitory effect on the proliferation of the PA-1 cell line. Bouhenna et al.
[4]
reported that chitin derivatives against human cancer cell lines RD and
Hep2. In the chitin was cytotoxic at IC50= 400 μg/ml against Hep2 cells lines. It concluded that the cytotoxicity is probably due to interactions among the negative charges groups of tumor cells and the positively charged groups of the chitin and chitosan. The same conclusion, as the previous authors, was given by Bouhenna et al.[4] who studied the cytotoxicity of chitin derivatives towards human cancer cell lines. In conclusion, the extracted chitin and chitosan were characterized by using FTIR, XRD and SEM analysis and the results revealed that the chitosan extracted from the exoskeleton of P. monodon is in alpha form. The findings of this study provide useful information for further studies to expand the biological applications of chitosan. Therefore, the results of this study that chitosan samples from P. monodon have significant anticancer properties against human ovarian cancer cell line, PA-1. Acknowledgements The authors are grateful thanks to the University of Madras, Department of Zoology for their help support in carrying out the present study. Reference 1. I. Younes, S. Hajji, V. Frachet, M. Rinaudo, K. Jellouli, M. Nasri, Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan, Inter. J. Biol. Macromol. 69 (2014) 489-98. 9
2. M.S. Benhabiles, N. Abdi, N. Drouiche, H. Lounici, A. Pauss, M.F.A. Goosen, N. Mameri, Protein recovery by ultrafiltration during isolation of chitin from shrimp shells Parapenaeus longirostris, Food Hydrocoll. 32 (2013) 28-34. 3. R. Salah, P. Michaud, F. Mati, Z. Harrat, H. Lounici, N. Abdi, N. Drouiche, N. Mameri, Anticancer activity of chemically prepared shrimp low molecular weight chitin evaluation with the human monocyte leukaemia cell line, THP-1. Inter. J. Biol. Macromol. 52 (2013) 333-339. 4. M. Bouhenna, R. Salah, R. Bakour, N. Drouiche, N. Abdi, H. Grib, H. Lounici1, N. Mameri, Effects of chitin and its derivatives on human cancer cells lines, Environ. Sci. Poll. Res. 22 (2015) 15579–15586. 5. H. Laribi-Habchi, A. Bouanane-Darenfed, N. Drouiche, A. Pauss, N. Mameri, Purification, characterization and molecular cloning of an extracellular chitinase from Bacillus licheniformis stain LHH100 isolated from wastewater samples in Algeria. Inter. J. Biol. Macromol. 72 (2015) 1117-28. 6. M.S. Benhabiles, R. Salah, H. Lounici, N. Drouiche, M.F.A. Goosen, N. Mameri, Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste. Food hydrocoll. 29 (2012) 48-56. 7. M. Dziril, H. Grib, H. Laribi-Habchi, N. Drouiche, N. Abdi, H. Lounici, A. Pauss, N. Mameri, Chitin oligomers and monomers production by coupling γ radiation and enzymatic hydrolysis, J. Ind. Eng. Chem. 26 (2015) 396-401. 8. Z. Limam, S. Selmi, S. Sadok, A. El Abed, Extraction and characterization of chitin and chitosan from crustacean by-products: Biological and physicochemical properties. African J. Biotechnol. 10(4) (2011) 640-647. 9. A. Khanafari, R. Marandi, S.H. Sanatei, Recovery of chitin and chitosan from shrimp waste by chemical and microbial methods. J. Environ. Health Sci. Eng. 5(1) (2008) 1-24. 10. W. Xia, P. Liu, J. Zhang, J. Chen, Biological activities of chitosan and chitooligosaccharides. Food Hydrocoll. 25 (2011) 170–79.
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11. R. Jayakumar, D. Menon, K. Manzoor, S.V. Nair, H. Tamura, Biomedical applications of chitin and chitosan nanomaterials - a short review. Carbohydr. Polym. 82 (2010) 227-232. 12. M. Dhanamani, S.L. Devi, S. Kannan, Ethnomedicinal plants for cancer therapy–a review. Hygeia J.D. Med. 3(1) (2011) 1-10. 13. C. Foster, D. Fenlon, Recovery and self-management support following primary cancer treatment. British J. Cancer 105 (2011) 21-28. 14. V. Shanta, R. Swaminathan, Population based cancer registry, Chennai cancer institute (WIA), Adyar, Chennai. Individual Registry write up, (2006- 2008) pp: 165-183. 15. M. Ogawa, E.L. Maia, A.C. Fernandes, M.L. Nunes, M.I. Oliveira, S.T. Freitas, Waste from the processing of farmed shrimp: a source of carotenoid pigments. Ciência e Tecnologia de Alimentos, 27 (2) (2007) 333-337 16. K. Gopakumar, Textbook of fish processing technology. Indian Council of Agricultural Research, New Delhi (2002) pp: 467-483. 17. L.A. Cira, S. Huerta, G.M. Hall, K. Shirai, Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery. Proc. Biochem. 37 (2002) 1359-1366. 18. H. K. No , S. P. Meyers, Preparation and characterization of chitin and chitosan: a review. J. Aqua. Food Pro. Tech. 4 (1995) 27-52. 19. K. Kurita, Chitin and chitosan functional biopolymers from marine crustaceans, Mar. Biotechnol. 8(3) (2003) 203–226. 20. M.S. Benhabiles, N. Abdi, N. Drouiche, H. Lounici, A. Pauss, M.F.A. Goosend, N. Mameri, Fish protein hydrolysate production from sardine solid waste by crude pepsin enzymatic hydrolysis un a bioreactor coupled to an ultrafiltration unit, Mat. Sci. and Eng. C 32 (2012) 922-928. 21. M.S. Blois, Antioxidant determinations by the use of a stable free radical. Nature 181 (1958) 1199-1200. 22. T. Mossman, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Method. 65 (1983) 55-64. 11
23. L. Sun, K. Feng, R. Jiang, J. Chen, Y. Zhao, R. Ma, H. Tong, Watersoluble polysaccharide from Bupleurum chinense DC: Isolation, structural features and antioxidant activity, Carbohydr. Polym. 79 (2010) 180-183. 24. A. Tolaimate, J. Debrieres, M. Rhazi, A. Alagui, Contribution to the preparation of chitin and chitosan with controlled physicochemical properties, Polymer 44 (2003) 7939-7952. 25. M. Kaya, T. Baran, A. Mentes, M. Asaroglu, G. Sezen, K.O. Tozak, Extraction and characterization of alpha-chitin and chitosan from six different aquatic invertebrates, Food Biophys. 9 (2014) 145-157. 26. M.T. Yen, J.H. Yang, J.L. Mau, Antioxidant properties of fungal chitosan from shiitake stipes. LWT-Food Sci. Technol. 40 (2007) 225-261. 27. M.K. Jang, B.G. Kong, Y.I. Jeong, C.H. Lee, J.W. Nah, Physicochemical characterization of α‐chitin, β‐chitin and γ‐chitin separated from natural resources, J. Polym. Sci. Part A: Polym. Chem. 42 (2004) 3423–3432. 28. G. Cárdenas, G. Cabrera, E. Taboada, S.P. Miranda, Chitin characterization by SEM, FTIR, XRD, and 13C cross polarization/mass angle spinning NMR. J. Appl. Polym. Sci. 93 (2004) 1876-1885. 29. M.T. Yen, J.L. Mau, Selected physical properties of chitin prepared from shiitake stipes. LWT-Food Sci. Technol. 40 (2007) 558–563. 30. M.T. Isa, A.O. Ameh, J.O. Gabriel, E. Leonardo, Extraction and characterization of chitin from Nigerian sources, J. Pract. Technol. 21 (2012) 73-81. 31. A. Kucukgulmez, M. Celik, Y. Yanar, D. Sen, H. Polat, A.E. Kadak, Physicochemical characterization of chitosan extracted from Metapenaeus stebbingi shells, Food Chem. 126(3) (2011) 1144-1148. 32. A.T. Paulino, J.I. Simionato, J.C. Garcia, J. Nozaki, Characterization of chitosan and chitin produced from silkworm chrysalides, Carbohydr. Polymer. 64 (2006) 98–103. 33. S. Matsugo, M. Mizuie, M. Matsugo, R. Ohwa, H. Kitano, T. Konishi, Synthesis and antioxidant activity of water-soluble chitosan derivatives. Biochem. Mol. Biol. Inter. 44(5) (1998) 939-948. 12
Fig. 1 FT-IR analysis of chitin/ chitosan from Penaeus monodon
13
Fig. 2 XRD analysis (A) Chitin and (B) Chitosan from Penaeus monodon
14
Fig. 3 SEM analysis for chitin (A) 500 µm and (B- C) 10 μm
Fig. 4 SEM analysis for chitosan (A) 40 µm, (B) 20 µm and (C) 50 μm 15
Fig. 5 Radical scavenging activity of chitin/ chitosan for Penaeus monodon
16
Fig. 6 MTT assay of chitin extraction from P.monodon against PA-1 ovarian cancer cell line (A) 10 µg/ml, (B) 20 µg/ml, (C) 30 µg/ml, (D) 40 µg/ml and (E) 50 µg/ml.
17
Fig. 7 MTT assay of chitosan extraction from P.monodon against PA-1 ovarian cancer cell line (A)10 µg/ml, (B) 20 µg/ml, (C) 30 µg/ml, (D) 40 µg/ml and (E) 50 µg/ml.
18