Fabrication and Characterization of Glucosamine Hydrochloride from Chitin of Horseshoe Crab Shell ( Tachypleus gigas )

. Preparation of glucosamine hydrochloride from the chitin of horseshoe crab shells using the chemical hydrolysis method has been done using HCl concentration variation ratios of 7%, 9%, 11%,14 % with a ratio of 9:1 (v/w) for 4 hours at a temperature of 90ºC. Determination of glucosamine hydrochloride characteristics was characterized using Fourier transform-infra red (FT-IR) spectroscopy, in which the characteristics of glucosamine hydrochloride obtained in the OH group of glucosamine hydrochloride were 3446 cm -1 (s), 3448 cm -1 (s), 3450 cm -1 (s), 3448 cm -1 (s), respectively. For NH group of glucosamine hydrochloride were 1557 cm -1 (s), 1559 cm -1 (s), 1556 cm -1 (s), 1560 cm -1 (s), respectively. For CN group of glucosamine hydrochloride were 1379 cm -1 (m), 1379 cm -1 (m), 1379 cm -1 (m), 1379 cm -1 (m), respectively. While the glycoside bond of glucosamine hydrochloride 1073 cm -1 (w), 1074 cm -1 (w), 1074 cm -1 (w), 1074 cm -1 (w), respectively. Determination of the concentration of glucosamine hydrochloride with Ultraviolet Spectrophotometer analysis at a maximum wavelength of 197 nm with a standard solution of N-acetyl glucosamine in a solution of phosphate acid 0.005%, in which obtained the concentration of glucosamine hydrochloride 7% = 33.67 ppm, 9% = 36.35 ppm, 11% = 40.16 ppm, 14% = 43.97 ppm.


Introduction
Glucosamine is an important building block for cartilage, which acts as a cushion at the ends of bones and prevents bones from cracking during movement.Glucosamine in the body keeps cartilage from calcifying faster.In addition, glucosamine is also useful for stimulating the formation and restoration of damaged cartilage tissue and helps reduce joint pain.Bones need calcium, while joints need glucosamine to be free to move and avoid joint pain.Bones that contain an adhesive substance and a little lime keep the hard bones from colliding with each other so that they can cause joint pain.
Normally, joint cartilage has a smooth surface and usually, there are small tears that can then be repaired by the body itself.But with age, these tears can no longer be repaired by the body because the ability to synthesize glucosamine decreases.In addition, with age, collagen cells also decrease.The body's ability to synthesize glucosamine decreases with age and this can lead to osteoarthritis and joint pain.Therefore, in order to keep the joint cartilage well lubricated, it is recommended to add glucosamine with external intake.This has caused the worldwide consumption of glucosamine to increase enormously, so reliable methods have been optimized for preparing high quality glucosamine products.
Glucosamine (2-amino-2-deoxyglucose, chitosamine) is an amino sugar obtained from the hydrolysis of chitin.Chitin is mainly produced from the outer shell of the crustaceans, arthropods, and fungi.In industry, glucosamine is produced by hydrolysis of the exoskeleton of crustaceans such as shrimp and crabs.Glucosamine products for people with arthritis are usually formulated as hydrochloride salts of glucosamine sulfate.
Several studies have been conducted on the hydrolysis of glucosamine.The Rotta Research Laboratory (1978) reported that glucosamine sulfate has been successfully prepared by immersing chitin in sulfuric acid solution, but this reaction produced a small amount of yield.In order to avoid this problem, Mojarrad, et al . (2006) stated that glucosamine sulfate can be prepared first by refluxing chitin with a hydrochloric acid solution to produce glucosamine hydrochloride 2 Materials and Methods

Preparation of Chitin
Preparation of chitin was conducted based on Agusnar, H . (2006).Firstly, shells were washed and then added 0.5% of NaOH for 24 hours.Next, washed with distilled water until the pH reached 6.7.A solubility test was carried out with 85% phosphoric acid.Demineralized with 5% of HCl solution for 24 hours, washed with H 2O to pH was 6.7.A solubility test was also performed with 37% of formic acid and dried at room temperature.Then mashed and sieved through an 80 mesh sieve.

The process of Hydrolysis of Glucosamine Hydrochloride
It was conducted using modified chemical hydrolysis from Mojarrad et al (2006).As much as 2.5 g of chitin powder was soaked in a hydrochloric acid solution with the same treatment at different concentrations of 7%, 9%, 11%, and 14%, with a ratio of 9:1 for 4 hours at 90ºC.Hydrolysis was continued by centrifugation of glucosamine hydrochloride slurry at 10,000 rpm for 15 minutes.The precipitate obtained was washed with ethanol, then centrifuged again at 10,000 rpm for 15 minutes.Furthermore, the precipitate obtained was dried in an oven at 40ºC for 4 hours.The glucosamine hydrochloride result was then analyzed for its characteristics, such as Fourier transform infrared (FT-IR) spectroscopy, and the concentration level was determined by Ultraviolet-Visible (UV) spectrophotometer analysis.

Analysis of glucosamine hydrochloride by FTIR
The glucosamine hydrochloride (7%, 9%, 11%, 14%) obtained and standard glucosamine were mixed with KBr in a ratio of 1:100 and then ground until smooth using a mortar.This mixture was placed in a press and pressed at a pressure of 800 kg.The pressed pieces were measured for absorbance through FTIR.The scanning range used is between 450 cm -1 to 4000 cm -1 .

Analysis of Glucosamine Hydrochloride with UV-Visible Spectrophotometer.
For phosphoric acid solution.
Phosphoric acid solution was made at 0.002%; 0.005%; 0.008%; and 0.012%, then the absorbance was measured at 197 nm in which distilled water was used as a blank.
For N-acetylglucosamine.
For Glucosamine Hydrochloride.
Solutions of glucosamine hydrochloride were (20%, 25%, 30%, and 37%) and standard glucosamine was prepared in 0.005% of phosphoric acid and then the absorbance was measured at 197 nm.

Chitin Preparation
Preparation of chitin was conducted based on Agusnar, H . (2006), and the result of chitin obtained was 30.8%.

Determination of Glucosamine Hydrochloride Characteristics by FTIR
The characteristics of glucosamine hydrochloride can be determined using FTIR analysis, which was shown in Table 1.

Sample
OH (cm -1 ) NH (cm -1 ) CN (cm -1 ) Glycoside Bonds (cm -1 ) Based on Table 1 shows that glucosamine hydrochloride from the hydrolysis of shellfish chitin with a ratio of variations in HCl concentration of 7%, 9%, 11%, and 14% produced an absorption band that was almost following the standard glucosamine hydrochloride.Mojarrad et al reported that it can be happened due to the influence of HCl concentration, temperature, and hydrolysis time so the glucosamine hydrochloride formed was still in a small concentration.
This can also be influenced by differences in chitin raw materials, because the chitin raw materials used were different, so resulting in different chitin content.

Determination of The Concentration of Glucosamine Hydrochloride By UV-Visible
Spectrophotometer Muzzarelli (1978) said that a good solvent for horseshoe crab was phosphoric acid, so phosphoric acid was used as a solvent in this study.The measurement of phosphoric acid absorbance can be seen in Table 2.  3. Table 3. shows that the concentration of phosphoric acid used was 0.005%.Muzzarelli (1978), the solubility of chitin was good at 0.005% of phosphoric acid, so 0.005% of phosphoric acid was used in this study.
The absorbance measurement of H 3PO4 (Table 2) shows an absorbance value was 1.18 of 0.005% of H 3PO4. Thus, the results of all measurements of the absorbance of Nacetylglucosamine and samples of glucosamine hydrochloride dissolved in 0.005% of H 3PO4 must be reduced by 1.18 to obtain the true absorbance of the two compounds.The results of the absorbance measurement of the standard N-acetylglucosamine in Table 3.
were plotted in a calibration curve so obtained in the form of a linear line.The regression line equation for this calibration curve can be derived using the least square method.

Figure 1 .
Figure 1.FT-IR spectrum of standard glucosamine hydrochloride

Tabel 3 .
Absorbance measurement data for N-acetylglucosamine in 0.005% of phosphoric acid at λ max = 197 nm