1. INTRODUCTION:
The attention towards polysaccharides of natural origin is constantly rising during the past decade. The natural polysaccharides are widely used in the field of food technology, cosmetics, pharmaceuticals and biomedical sciences. Exploitation of new sources of polysaccharides of different origin is well documented in the literature1. They exhibit good mechanical properties and are widely used as fibers, films, adhesives, rheology modifiers, hydrogels, emulsifiers and drug delivery agents. Sodium alginate (SA), xanthan gum (XG), guar gum, scleroglucan, and locust bean gums are some of the natural polysaccharides which are fueling the interest of the researchers dealing with the development of drug delivery systems2.
The functional groups of polysaccharides have been explored for chemical modification to change their properties like solubility, swelling, viscosity, and degradation3.
Tamarind seed contains approximately 65% of the gum and it may be used for the development of specific drug delivery systems. The polysaccharide that is present in tamarind gum (TG) is known as tamarind seed polysaccharide4. Functionalization of gum into Carboxymethyl tamarind gum (CMTG) may improve physicochemical properties of tamarind gum. Despite being well suited for pharmaceutical application, TG exhibits some potential drawbacks. TG has a dull color and unpleasant odor. Its insolubility in water and degradation in aqueous environment has forced the scientists to chemically modify its functional groups5. Various modifications which have been executed till date include carboxymethylation6, acetylation7, hydroxyl-alkylation8 and thiolisation9,10. Such modifications have caused alteration in the solubility, viscosity, swelling, and stability of tamarind gum. TG and CMTG has been used in the development of various drug delivery systems11–16. Considering above facts there is demand to explore physicochemical properties of TG in order to ensure its suitability as an excipient in development of drug delivery systems. Hence, an attempt was made to extract, modify and characterize tamarind gum.
2. MATERIALS AND METHODS:
Tamarind kernel powder was kindly gifted by Chhaya Industries, Barshi, Maharashtra (India). Tamarind seeds were purchased from local market. All other chemicals and solvents were supplied by Loba Chemie, Mumbai, Maharashtra (India).
2.1 Extraction of TG:
TG was extracted from the tamarind kernel powder available in the market. The 20 g of defatted tamarind seed powder was added to 200 ml of cold distilled water to prepare slurry. Slurry was then poured into 800ml of boiling distilled water containing citric acid (0.2 %). The solution was boiled for 20 min with stirring in a water bath. Resulting thin clear solution was kept overnight (24 h) so that most of the proteins and fibers settle out, following which the solution was centrifuged at 5000 rpm for 20 min. Supernatant liquid was separated and poured into the excess of absolute alcohol with continuous stirring (1:1). Precipitate was washed with 200 ml of absolute ethanol, diethyl ether, and petroleum ether and/or acetone and dried at 50-60°C for 10 h. Dried polymer was powdered, sieved and stored in a desiccator until further use17,18. Also, TG was extracted using tamarind seeds. Percent yield was calculated and recorded. Flow chart of TG extraction is given in Figure 1.
2.2 Characterization of TG:
Organoleptic evaluation of TG:
Separated gum was evaluated for color, odor, taste, fracture, and texture. TG was cream brown in color, odorless and tasteless with irregular in shape. TG was rough in touch, texture, and fracture.
Shape of TG particles:
TG particles were observed under the Motic microscope at 10X resolution.
Identification tests:
Identification tests for TG were performed as per the standard procedures.
Determination of solubility:
A 100 ml (1% w/v) suspension of polysaccharide was transferred into a blender jar and blended at low speed for 3 min. The suspension was transferred to a centrifuge tube and centrifuged for 15 min. A 50 ml aliquot of supernatant was taken into a pre-weighed petri plate and dried in hot air oven at 105°C until the constant weight was obtained. Percent cold water solubility was then calculated and recorded19.
Determination of PH:
A weighed quantity of TG was dispersed in distilled water to get 1% w/v solution. The pH of the resultant solution was measured by using pH meter.
Figure 1: Extraction of TG.
Swelling index of TG:
Swelling profile of TG was determined by transferring accurately weighed 1 gm of TG into separate 25 ml measuring cylinders. The volume of each of cylinder was adjusted with solvent and observations were recorded for an increase in the volume of TG. Readings were taken at specified times until a constant volume was observed in each of the cylinders. The study was performed in triplicate4.
1
Determination of viscosity:
Accurately weighed (1 gm) required quantity of TG was transferred to into separate 100 ml volumetric flask. These were made up to mark with distilled water. After one hour viscosity was measured. Small sample adapter (7ml) was used for measurement of viscosity with Spindle No. 21 rotated at 100 RPM. The study was performed in triplicate.
Bulk and tap density:
Accurately weighed 100 gm TG transferred into 250 ml graduated measuring cylinder and the initial volume of powder was recorded as V0. The powder was subjected to 300 taps/min in tap density apparatus (Electrolab, Mumbai). The tapped volume was recorded as Vf. Bulk and tap density of TG were calculated using the following equations:
2
3
Carr’s Index and Hausner’s ratio:
Carr’s index and Hausner’s ratio are measures of the relative importance of interparticulate interactions. The Carr’s index gives an idea about flowability of the powder. Carr’s index can be calculated using the following equation:
4
Hausner’s ratio was calculated using the equation below:
5
ATR-FTIR study of TG:
Infrared spectrum of was obtained using ATR-FTIR spectrophotometer (Shimadzu, Miracle 10, IR Affinity, Japan). The samples to be analyzed were placed onto the ATR and spectra were recorded in the range of 600–4000 cm−1 at an average of 25 scans and resolution of 4 cm−1.
Thermal analysis of TG:
Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of TG was performed using Mettler-Toledo TGA/DSC1 thermogravimetric analyzer (Mettler-Toledo, Switzerland). Samples were heated from 30oC–300oC at the rate of 10oC/min, under a nitrogen atmosphere (flow rate: 10 ml/min).
Solid state 13C NMR spectroscopy:
Solid state 13C cross-polarization-magic angle spinning (13C CP-MAS) NMR spectrum of TG was measured using JEOL-ECX400 spectrometer operating at 400 MHz (contact time of 3.5 ms, a relaxation delay of 5s, sweep width of 35 kHz and spinning speed of 10KHz). The chemical shifts were calibrated with the external hexamethylbenzene standard methyl resonance at 17.3 ppm.
X-ray powder diffraction:
X-ray diffraction (XRD) patterns of TG and CMTG were recorded using X-ray diffractometer (PW1729, Philips, The Netherlands) with a copper target, operated at voltage of 30 kV, 30 mA current, at 2°C/min scanning speed and scanning angle ranging from 0 to 90° (2θ).
2.3 Synthesis of CMTG:
Carboxymethylation of TG was carried out using the method reported by Goyal et al., 200720 (see Figure 2). TG (0.05 mol) was dispersed in 80ml alkaline aqueous methanol (0.158 mol sodium hydroxide). To this dispersion monochloroacetic acid (0.09 mol) was added in solid form with continuous stirring for 15 min. The flask was immersed in a thermostatic water bath and the temperature was maintained at 70°C for 60 min. The contents of the flask were shaken occasionally during the course of the study. The reaction product was filtered, dissolved in water and neutralized with dilute acetic acid. The reaction product was precipitated in ethanol and washed twice with aqueous methanol (80 %, v/v) followed by pure methanol. The product was initially dried at room temperature and then in a vacuum oven at 40°C for 4 h to obtain CMTG. The degree of substitution of CMTG was determined by titrimetric method9,21.
Figure 2: Synthesis of CMTG.
2.4 Characterization of CMTG:
Organoleptic properties, Shape of particles, identification tests, solubility, pH, swelling index, viscosity and powder characteristics of CMTG was as per procedure given in evaluation of TG. Further carboxymethylation of TG was confirmed by ATR-FTIR, thermal analysis and solid-state 13C NMR of CMTG.
3. RESULTS AND DISCUSSION:
3.1 Extraction of TG:
Results of extraction of TG are given in Table 1. Initially, TG was extracted from the tamarind seeds. The process to collect gum from tamarind seed was tedious, time-consuming and yield was also very low (less than 20%)4. This might be due to wastage of gum during extraction. Tamarind kernel powder is available in the market which contains fats, proteins, and carbohydrate. Fats present in tamarind kernel powder were removed with petroleum ether and was further used for extraction of TG. Tamarind kernel powder was added to boiling an aqueous solution of citric acid helps to separate proteins from the TG due to precipitation. TG present in the supernatant solution was separated by alcohol-precipitation18. The yield of TG was found to be more than 50% (~52.89±3.31%) when tamarind kernel powder was used for extraction. Obtained TG was passed through Sieve No 80 and stored in desiccator until further use.
Table 1: Extraction of TG
|
Parameter |
Tamarind seeds |
Tamarind kernel powder |
|
Weight of Raw material (g) |
20 |
20 |
|
Yield (g) |
3.54 |
10.47 |
|
Yield (%) |
17.69 |
52.89 |
3.2 Characterization of TG:
Organoleptic evaluation of TG:
Separated gum was evaluated for color, odor, taste, fracture, and texture. TG was cream brown in color, odorless and tasteless with irregular in shape. TG was rough in touch, texture and hard.
Shape of TG particles:
TG powder was observed under a Motic microscope at 10X resolution. Shape of particles is shown in Figure 3.
Figure 3: Microscopic image of TG.
TG particles were found to be irregular in shape with a rough surface. Most of the particles were of rectangular in shape. This may be due to the formation of thread-like structure during the extraction process.
Identification tests:
Results of identification tests are given in Table 2. Identification tests showed the presence of carbohydrate in the TG powder. When TG was mixed with Molisch’s reagent followed by addition of sulfuric acid the violet color ring was appeared at the junction of the mixture in a test tube which confirms the presence of carbohydrate. TG powder showed negative test results for alkaloids, tannins, proteins, fats, and mucilages. This can be considered as proof for the purity of the isolated TG and free from proteins and fats.
Table 2: Chemical characterization of TG
|
Test |
Present (+) /Absent (-) |
|
Carbohydrate |
+ |
|
Hexose sugar |
+ |
|
Monosaccharides |
- |
|
Alkaloid |
- |
|
Tannins |
- |
|
Fats and oils |
- |
|
Proteins |
- |
|
Amino acids |
- |
|
Mucilages |
- |
Determination of solubility:
Cold water solubility of the TG sample was found to be 1.59±0.16 mg/ml. When the sample was heated it forms viscous solution due to swelling of TG in water. It indicates TG may form a gel. TG powder was found to be insoluble in ethanol, methanol, benzene, ether, and acetone.
Determination of pH:
pH of 1% TG in distilled water was found to be 6.52±0.18. It indicates that the TG is slightly acidic in nature.
Swelling of TG:
Swelling of TG was found to be 1.6 times of the dry volume of gum. It indicates TG can be used in sustained or controlled drug delivery of drugs.
Determination of viscosity:
Viscosity of 1 % TG was found to be 38.53±2.21 cP. It indicates a high concentration of TG is required to produce a gel.
Powder characteristics of TG:
Micromeritic properties of TG powder are given in Table 3. The flow properties of the powder material are dependent on the shape of the particles. It indicates that the prepared TG may be suitable for the development of solid dosage forms with the addition of suitable glidant.
Table 3: Micromeritic properties of TG
|
Parameter |
Value |
|
Bulk density (g/ml) |
0.48 |
|
Tap density (g/ml) |
0.57 |
|
Carr’s Index (%) |
15.8 |
|
Hausner’s ratio |
1.19 |
|
Angle of repose (°) |
31.4 |
ATR-FTIR study of TG:
ATR-FTIR spectrum of TG is given in Figure 4. ATR-FTIR spectrum of TG exhibited broad strong peaks at 3500–3000 cm-1 belonging to stretching vibration of –OH groups present in glucose, xylose and galactose units in the polysaccharide. A strong peak at 1039 cm-1 and 1143 cm-1 are attributed to the C-O stretching vibration of alcoholic group. The medium peak at 2920 cm-1 belonged to asymmetric stretching of CH. The peaks at 1747 cm-1 and 1689 cm-1 were due to carbonyl (-HC=O) stretching22.
Thermal analysis of TG:
TGA-DSC of TG is given in Figure 5. Thermal decomposition curve of TG showed two main stages of decomposition. The first stage begins at 35°C and ends at 100°C. This may be due to the removal of free and bound water from the polymer. The second stage of weight loss was observed around 228°C to 300°C with 35% loss of weight. DSC thermogram of TG showed endotherm at 238.56°C. DSC curve supports the weight loss as evident in TGA curve.