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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304 Vol.9, No.1, pp 171-178, 2016
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Study on the Bioactive Compounds of Shark
(Prionace glauca) Cartilage and its Inflammatory Activity
T.I.Agustin1, Wahyu S.1, and E. Yatmasari2
1Faculty Engineering and Marine Science, Hang Tuah University, Indonesia
2Faculty of Medicine Hang Tuah University, Indonesia
Abstract: The objective of the study was to identify the bioactive compounds of shark (Prionace glauca) cartilage and observe its antiinflammatory activity. This study was descriptive and used laboratory experiment separated into 3 phases: preparation and extraction, bioactive compound identification using Fourier Transform Infra Red (FTIR) spectroscopy, and in vivo antiinflammatory activity test using wistar rats. Results showed that IR-spectra of chondroitin and glucosamine isolated from the shark cartilage had very similar absorption peak to the standard glucosamine sulphate and chondroitin sulphate. The IR-spectra of standard chondroitin possessed strong absorption peak at the wavelength of 1627.87 cm-1 and 1413.72 cm-1 indicating that the presence of carboxyl groups with amine and sulphate. The mean percent of inflammatory inhibition was 13.40%, 4.02%, 4.15%, 3.88 % and 2.01 %, respectively, for indomethacine, shark cartilage powder,chondroitin extract, glucosamine extract and the combination of chondroitin-glucosamine extracts. The extract of chondroitin suphate had higher inflammatory activity than that of other treatments, but not significantly different from that of shark cartilage powder.
Keywords: Shark cartilage, anti-inflammatory, in vivo.
Introduction
Indonesia is a maritime country of rich biopharmaceutical products. Sharks are one of the fishes possessing high number of species and believed to have compounds potential to anti-cancer. It has been used in either conventional form or in modern pack. The use of shark cartilage as medicine in Indonesia is uncommon yet. The fish is fished for their fins as delicious soup dish, while the flesh and cartilage are often wasted. Information on bioactive compound purificatiom and anti-angiogenic activity will increase the economic value of sharks1.
Shark cartilage is composed of collagen, amino acid, mineral, and glycosaminoglycan. It contains more than 40% of pure collagen proteins plus essential amino acids and non essensial amino acids. Collalagen and amino acids are required for tissue elasticity, fissured skin, ligament, and tendon healing, and growth and rejuvenation of skin and connective tissues2. Previous study3 revealed that shark cartilage held 30.74% proteins and 10,673.59 ppm of calcium. Total content of amino acid was 23.66% of protein, but its amino acid composition was higher than PAP (Provisional Amino Acid Pattern). Glucosamine and chondroitin are amino glucose as major components of shark cartilage. Both compounds act to stimulate the formation of glycosaminoglycan, proteoglycan, and hyaluronic acid. Clinical studies have revealed that glucosamine helps reducing arthritis symptomes. In short period, glucosamine has been tested and demonstrated effective symptoms osteoarthritis patients. Glucosamine could also take protective role against modification disease in osteoarthritis case by providing anti-cathabolic effect and anti-inflammatory activity on chondrocytes4-6. This study was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage usingstudy was aimed at identifying the bioactive compounds of shark cartilage using
Materials and Method
Materials
Shark cartilage samples were obtained in frozen condition from frozen fish processing company of “CV Angin Timur”. These were taken to Fisheries Product Processing Laboratory of Hang Tuah University, Surabaya, using cool boxes. The frozen samples were melted in flowing water and cleansed from flesh remnants attaching to the cartilage and non-cartilaginous tissues. The cartilage was then stored at -20oC up to extraction. All chemicals used were pro-analytical quality, such as ammonium carbonate, glacial acetic acid, indomethacin, carrageenan, ether, and Na-CMC. For antiinflammatory activity test, wistar-strained rats were employed.
Equipment
This study used knife, thermometer, dryer, pan, blender, a 80-mesh screen, plastic clip, cuvet, magnetic stirrer, centrifugator, freeze dryer, IR-spectrophotometer, marker, pH pen, rat cage, rat drinking water supply, measuring tube, incubator, stirrer, sonde, and plethysmometer.
Test Animals
Male wistar rats, Rattus novergicus, of each 100 – 120 g BW were obtained from CV. Surabaya Mouse Service. They were kept in cages at room temperature. Drinking water and feed were given ad-libitium. They were then separated into 6 groups each of which was 4 rats. Two groups were employed as positive and negative controls. The former was orally induced with indomethacin of 10 mg/kg BW, and the latter with that of 1 ml of 2% Na-CMC. Other 4 groups were treatment groups that were daily administered with cartilage powder, extracted glucosamine, extracted chondroitin, and the combination of glucosamine-chondroitin extract each of which with the dose of 100 mg/kg BW. The test animals were previously acclimated for 2 weeks to adapt to the laboratory conditions. All research procedures have followed the guidance and the regulation of the ethical clearance committee of Hang Tuah University.
Bioactive compound extraction
Frozen shark cartilage samples were melted at 4oC, then chopped (about 1 mm3 size), dried, milled, and sieved through a 80-mesh sieve to obtain fine powder. The cartilage powder was then dissolved in 0.1 M ammonium carbonate pH 8 (10 gr of cartilage powder per 100 ml of ammonium carbonate), agitated with magnetic stirrer for 24 hours at room temperature, cengtrifuged, and the supernatant was dried in a freeze dryer to gain glucosamine compound7. Chondroitin extraction was done through incubation of 10 g of shark cartilage powder in 100 ml of pH 4.5 water at 37oC for 7 hours, pH is controlled through acetic acid addition. This condition is an optimal treatment for chondroitin extraction of cow’s nose cartilage8. After incubation and then centrigugation, the supernatant was dried in a freeze dryer for 12 – 18 hours to obtain chondroitin compound.
Bioactive Compound Identification with FTIR Spectrocopy
FTIR spectroscopy is an established and continuously growing analytica tehnique that enables to do simple, reliable, and fast analyses of various types of samples9. IR spectrocopy is highly useful for qualitative analysis (identification) of organic compounds since each organic compound produces unique spectrum with suitable structural peak for different features. Each functional group also absorbs the infra-red light at the unique frequency. For instance, a carbonyl group, C=O, always absorbs the IR light at the wavelength of 1670 cm-1 to 780 cm-1, that causes the carbonyl bond stretchable10. A chemical bond can vary with its energy level by giving specific frequency. This has become the principle of infra-red spectroscopiy measurement, in which if the sample is exposed to infra-red light the functional group of the sample will absorb the IR radiation and vibrate through stretching, bending, forming or a combination of vibration. The absorption/vibration can directly be correlated with chemical (biochemical) types, and the spectra of IR absorption can be illustrated as characteristics of chemicals or biochemicals11.
In Vivo Anti-inflammatory Test
In vivo anti-inflammatory test followed the method of Winter et al. (1962)12. This study used 24 inviduals of male wistar rats divided into 6 groups each of which held 4 individuals. Before the experiment started, carrageenan had been prepared by dissolving 0.5% carrageenan in aquadest (w/v). Each animal was subcutaneously injected with 0.1 ml of 0.5% carrageenan in the rear right foot after the rat foot had been measured as initial data (v0). The intumescence was measured after 1, 2, 3, 4, and 5 hours of carrageenan injection. The intumescence volume of the rat foot was measured using a plethysmometer, and the percent of the intumescence from inflammation was calculated as follows:
% Volume of edema , and then
percent inhibition of edema was calculated as follows:
% Edema inhibition
where
Vt : Volume of rat feet at t
Vo: Volume of rat feet at to
R : Mean inflammation of negative control group
S : Mean inflammation of treatment groups
Results and Discussion
Bioactive Compounds of Shark Cartilage
Infra-red spectral absorption was read at the wavelength of 400-4,000cm-1. Glucosamine isolated from the shark cartilage possesses nearly similar functional group to standard glucosamine, glucosamine sulphate. The IR spetra of shark cartilage powder and glucosame powder of shark cartilage are presented in Fig. 1 and 2.
Figure 1. FT-IR Spectra of Shark Cartilage Powder
Figure 2. FT-IR Spectra of Glucosamine Isolated From Shark Cartilage
Fig. 2 demonstrates that the IR spectra of glucosamine isolated from shark cartilage is indicated with wide band above ther wavelength of 3000 cm-1 and strong absorption at the wavelength of about 1,650 cm-1 and 1,050 cm-1. Hydroxide functional group is dominat group and possesses wide and strong IR absorption characteristics at the wavelength of 3,423.41 cm-1 dan 3,448.49 cm-1. The asymetrical NH group has the IR absorption characteristics at the wavelength of 1,662.62 cm-1 and 1,627.81 cm-1, while the symetrical NH group occurs at the wavelength of 1,454.23 cm-1 and 1,404.00 cm-1. The stretching and deformation of –C-O-H bond vibration occurring at the wavelength of 1,627.81cm-1 and 1,454.23cm-1 are corresponding to the presence of carboxylate association with amine and sulphate. It indicates that glucosamine isolated from the shark cartilage is classified as glucosamine sulphate. Exogenous glucosamine generally consisted of glucosamine hydroxide and glucosamine sulphate. Ini Japan, glucosamine sulphate is an osteoarthritis drug, not nutritional supplement13. The infrared spectrum of standard chondroitin sulphate chondroitin isolated from the cartilage can be seen in Fig. 3 and 4.
Figure 3. FT-IR Spectra of Standard Chondroitin Sulphate (Manjusha, 2011)
Figure 4. Chondroitin Isolated From Shark Cartilage
The IR-spectrum of chondroitin isolated from the shark cartilage (Fig. 4) is compared with the standard IR-spectrum of standard chondroitin sulphate (Fig. 3). In the case of standard chondroitin sulphate C, stretching and deformation of –C-O-H- bond vibration at the wavelength of 1,637.63 cm-1 and 1,420.03cm-1 is corresponding to the carboxylate association with amine and sulphate, while the peak intensity of chondroitin isolated from shark cartilage occurs at the wavelength of 1,661.31cm-1 and 1,415.05 cm-1 (Fig. 4). The wide band above the wavelength of 3,000cm-1 determines the stretch of hydroxyl vibration of polysacharride and water involving hydrogen bond. Sulphate bond of standard chondroitin sulphate (Fig. 3) started from the wavelength of 1,266.78 cm-1 to 829.06 cm-1. The same peak intensity was found at the wavelength of 1,233.21cm-1 to 783,05cm-1 in chondroitin isolated from shark cartilage indicating sulphate group. Chondroitin sulphate is particularly useful for taking care of cartilage structure and function, ostearthritis joint pain, and anti-inflammatory activity14.
Antiiflammatory activity test
Carrageenan is a suitable model for acute inflammatory study of rats15,16. Foot intumescence model from carrageenan induction is very sensitive to evaluating orally antiinflammatory agent, particularly natural products, and highly reliable for acute antiinflammatory studies17. Subcutaneous injection of carrageen in the rat foot causes edema formation that is a biphasic event18,19. Phase 1 (0 – 2.5 hours after carrageenan injection) is mainly caused by inflammatory mediator release, such as histamine, serotonin, from surrounding damaged tissues. Phase 2 (3 – 6 hours after carrageenan injection) is supported by releasing prostaglandin produced by the macrophage and mediated by bradykinin and leukotrienes17. In vivo anti-inflammatory activity was observed once an hour for 5 hours of post-carrageenan induction. The effect of the bioactive compounds isolated from shark cartilage on the rat foot induced with carrageenan is given in Fig. 5 and the anti-inflammatory activity in Table 1 .
Figure 5. The effect of shark cartilage bioactive compounds on the carrageenan-induced rat foot.
Fig. 5 shows that maximum edema volume occurs in the third hour. It is in line with the previous study that edema grows fast in 3 hours of post-carrageenan induction and stays at the maximum volume for about 5 hours after induction20. The administration of shark cartilage powder, glucosamine isolate, chondroitin isolate, and glucosamine and chondroitin isolate combination gives similar effect to that of indomethacin application, edema reduction at the third hour and reaching the initial volume at the fifth hour.
In pharmacokinetics, the ability of compound to reduce edema of the test animal foot from carrageenan injetion is expressed as inflammatory ability21. This is useful to determine whether two similar dose formulations can release the same dose in the body. The value of antiinflammatory ability is obtained by comparing the area below the edema curve volume of the treatment animal with the area below the negative control curve. The area under the curve (AUC) is the area below the curve of drug concentration in the plasm plotted against time. The AUC formula is as follows:
AUC0-5 =
and the anti inflammatory ability was calculated as follows:
Anti inflammatory ability =
where
AUC : Area under curve
AUCx : Area under edema percentage curve against time in average negative group
AUCp : Area under the percent edema curve against time in average treatment group
V0 : Initial volume (before induction)
V1-5 : Edema volume of t1 – t5
0-5 : Measurement time of edema volume (t0 – t5)
t1-5 : Measurement time
Table 1. Data of AUC and Anti inflammatory Ability
Treatment Group |
AUC |
Anti inflammatory (%) Ability |
Negative Control: Na-CMC 2% |
0.373 |
|
Positive Control: Indomethacin |
0.323 |
13.40 |
Test I: Cartilage Powder |
0.358 |
4.02 |
Test II: Chondroitin |
0.3755 |
4.15 |
Test III: Glucosamine |
0.3585 |
3.88 |
Test IV: Combination glcosamine and chondroitin |
0.3655 |
2.01 |
Table 1 demonstrates that the highest anti-inflammatory ability occurs in rat treated with indomethacin (positive control). Indomethacin is non-steroid indol derivative anti-inflammatory drug which is chemically 1-(p-chlorobenzoil)-5-methoxy-2-methyl-indola-3-acetic acid. Indomethacin works more effectively than aspirin or other non steroid antiinflammatory drugs, is the strongest prostaglandin synthetic inhibtor. It is well absorbed after oral administration and partly related with plasm protein (about 90%). Metabolisms occur in hepar in unchangable form, and this drug is excreted into the bile and urine22. Shark cartilage23 contains hialuronic acid, a compound responsible for the viscosity of synovial fluid and synthesized by synovial membrane cells. The liquid part of synovial fluid is believed originating from transudate. The synovial fluid acts also as source of nutrition for joint cartilage. It was also found24 that as glycosaminoglycan, hialuronic acid worked as wound healing as well.
Glucosamine is a basic component of glycoproteins. It is known to be able to reduce the cartilage proteolytic process and reform the damaged glucosaminoglycan. In vitro study demonstrated that glucosamine had antiinflammatory effect through inhibition of inflammation-nediating cell production. This effect was demonstrated in experimental rats with arthritis25. Chondroitin sulphate is a derivative of cartilage components, either from shark, cow (bovine cartilage) or chicken. Previous studies in France and Italy in 1998 found that oral or injection administration could help increasing the joint movements and reducing the joint pain26.
Conclusion
The value of anti-inflammatory ability shows the presence of anti inflammatory effect of shark cartilage powder, chondroitin isolate, glucosamine isolate, and the combination of glucosamine–chondroitin. The highest percentage of the anti-inflammatory ability occurs in chondroitin test group and nearly similar to that of the cartilage powder. Chondroitin isolate production is more difficult and needs higher cost, so that the use of the cartilage powder is more effective and efficient.
Acknowledgment
This great appreciation is given to DP2M - Dikti – Depdiknas that provided this research grant through the decree numbered 027/SP2H/PDSTRL/K7/KL/II/2013. February 15th, 2013. In addition, we also thank to Junita Cahyati for her great help in this work.
References
www.scholarsresearchlibrary.com . accessed on October 1st, 2012.
http:// angsukri. blogspot.com/ 2011/07/muskuloskletal.html. Accessed on October 21, 2013.
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