1. INTRODUCTION:

In the recent years, it has been observed that the researchers is moving towards the utilization of herbal products as active ingredients for curing and treating of many classes of disease includes cancer, asthma and various hypersensitive reactions. Since, these products show easy availability and low risk of toxicity due to biodegradable in nature after consumption. Data evidence that market potential of natural products are continuously increasing day by day.

Reports show that almost 50% of drugs approved since 1994 are based of naturally occurring or derived products [1]. Increased demand of herbal cosmetics and toilet preparations show the potential of herbs in cosmetology also [2]. Recently, a new role of herbal product has been identified as processing excipients for effective and safe delivery of pharmaceuticals. A variety of herbal materials such as gum [3], mucilage [4], pectin [5] and starch [6] have been reported for successful delivery of medicaments. It would be logical to say that any polysaccharide should be undergone various evaluator parameters before its processing. With the same context, in the present study, an attempt was made to investigate the physical, rheological and biological behavior of two selected natural plants polysaccharide. Polysaccharides were isolated from seeds of Trigonella foenum graecum, TFG (Fenugreek, family Fabaceae) and Tamarindus indica, TI (Tamarind family Fabaceae). Polysaccharide of TFG is nonionic, neutral, branched structure consist of cellulose like backbone which carries xylose and galactoxylose subunits. Whereas, TI polysaccharide consist of five components such as kaempferol 3-O-β-d-glucopyranoside, kaempferol 7-O-glucoside, kaempferol 3-O-α-l-rhamnosyl (1→2) β-d-xyloside, kaempferol 7-O-β-d-glucopyranosyl (1–4) β-d-glucopyranoside and kaempferol 3-O-β-glucosyl (1→2) (6′-O-acetyl)-β-d-galactoside [7]

2. MATERIAL AND METHODS:

Seeds of and Trigonella foenum graecum and Tamarindus indica were purchased from local market of Kanpur (UP). All other chemicals used in extraction and characterization of polysaccharides were of analytical grade and were procured from commercial sources.

2.1. Polysaccharide Extraction:

Polysaccharide was extracted as per reported methods in various publications [8]. An accurate quantity (200 g) of seeds of TFG and TI were washed with double distilled water to remove any adherent material. The TI seed were process by separating the brown peels from the kernel seed with the blander and plastic sieve were used to separate the seed. Then, the seeds were crushed lightly and about three volumes of water were added in both seed separately. Both content were heated at 60°C on a water bath for about 4 h until the slurry was prepared. The viscous solution was then filtered and the filtrate was diluted with three volumes of water and kept undisturbed overnight in a refrigerator, so that most of the undissolved portion settled down. After this the clear supernatant portion was decanted and concentrated at 60±1°C in a rotary vacuum evaporator. The concentrate was cooled to room temperature and precipitated in about three volumes of acetone. The precipitate was washed repeatedly with acetone and dried at 50±1°C. Afterwards, the protein contaminations were removed by Savage method. The dried material was ground by a mechanical grinder and kept in a desiccator till further use.

2.2 Polysaccahride Characterization:

2.2.1. Organoleptic and Micrometric Evaluation:

Extracted polysaccharides of TFG and TI were evaluated for organoleptic properties such as colour, odour and taste. In order to determine the pH of the both polysaccharide, 1% w/v solution was prepared in distilled water. The pH of the both solution was measured using digital pH meter (pH tutor, Eutech). Bulk density of a powder is the ratio of mass of untapped powder sample and its volume including the contribution of the inter-particulate void volume. The bulk density of powder depends mainly on the particle size, particle shape and the trend of particles to adhere to one another. Bulk density of TFG and TI polysaccharides powder were determined by measuring the volume of a known weight of powder sample that may have been passed through a sieve 25 into a graduated cylinder. Mathematically bulk density can be represented by equation 1.

Bulk density= Weight of powder / Bulk volume (1)

Tapped density is determined by mechanically tapping a graduated measuring cylinder containing a powder sample. Tapped density of TFG and TI polysaccharide powder was determined by placing a graduated cylinder into a mechanical tapper apparatus (Electrolab, U.S.P). After observing the volume, the cylinder was mechanically tapped for 100 times or until the powder achieves a constant volume. Mathematically tapped density can be represented by equation 2.

Tapped density= Weight of powder/ Tapped volume (2)

Angle of repose was determined by using funnel method (by keeping a funnel vertically in a stand at a specified height above a graph paper placed on a horizontal surface). Approximately 2 g of polysaccharide powder was transferred into the funnel keeping the orifice of the funnel blocked by the thumb. Then the orifice of the funnel was opened to release the powder on the paper to form a smooth conical heap. The radius of the heap (r) and the height of the heap (h) were measured. Angle of repose can be represented by equation 3.

Angle of repose (θ) = tan^-1height/radius (3)

Compressibility index is used as an important parameter to determine the flow behaviour of the powder. It is indirectly related to the relative flow property rate, cohesiveness and particle size. It is simple, fast and popular method for predicting flow characteristics. Carr’s index can be represented by equation 4.

tapped density-bulk density

Carr’s index(%)=-------------------------------------x 100

tapped density (4)

2.2.2. Surface Characteristics of Polysaccharide using Scanning Electron Microscopy (SEM):

SEM is one of the advanced technologies which are used to determine the surface characteristics of a material. To study the surface characteristics of both extracted polysaccharide, SEM technique was used. The dry powder of TFG and TI was placed on an aluminum stub with double sided adhesive tape and evaporated with carbon and then sputtered with gold to make the samples electrically connected. Carbon was layered to a thickness of approximately 10 nm and gold was layered to approximately 25 nm. The photographs were taken at different resolution by using SEM apparatus (Leo, 435 VP).

2.2.3. Determination of Glucose in Extracted Polysaccharide:

Total glucose content in both extracted plant polysaccharide was estimated by using Pheno-sulfuric acid method describe by Dubois et al.1956 [9]. Briefly, Two milliliters of standard grade sugar solutions (ranging from 10 and 100 μg/ml concentrations of sugar) were prepared in distilled water. Similarly, test solutions of both polysaccharide (TFG and TI) were prepared by dissolving 200 µg into 2 ml of distilled water. All standard and test solution were transferred into test tubes. To these solutions 1 ml of 5%w/v phenol solution and 5 ml of concentrated sulfuric acid was added. All the mixture were shaken vigorously and equilibrate at 30^oC till estimation. The absorbance of the characteristic yellow-orange color was measured at 488 nm by using UV-visible spectrophotometer (Shimadzu 1700, Japan). Blanks were prepared by substituting distilled water for the sugar solution. Sugar content in both test solution were calculated in comparison to the standard curve of sugar.

2.2.4. Molecular Weight Determination:

Molecular weight of both extracted polysaccharide was determined by equation 5, as reported in publication by Kar et al. [10].

1/ M_w,ave = lim_c→0(HC/τ) (5)

Where M_w,ave is the average molecular weight (kg/kg mol), τ is the turbidity of solution (m^-1) which was measured by Nephlo-Turbidity Meter (Systronic, India). H can be calculated by equation (6) given by Allock and Lampe [10]

H = 32π³n_o²/3λ⁴N_o (n-n_o/C)²(6)

Where n_othe refractive index of the solvent (0.1 M sodium phosphate buffer, pH 7.0), n the refractive index of the polysaccharide solutions, λ wavelength of light (0.5893x10^-6m), N_o avogardo number (6.023 x 10²³) and C is the concentration of polysaccharide solution (2.5, 5, 10, 15 and 20 kg/m³). The refractive index of polysaccharide solutions (TFG and TI) were determined by refractometer using sodium vapour lamp. The intensity of light scattered through polysaccharide solution was measured as percentage of light transmitted through solutions, as compared to that through 0.1M sodium phosphate buffer. M_w,ave was determined by plotting a graph between HC/τ versus the concentration of TFG and TI solutions.

2.2.5. Rheological Characterization:

The R/S-CPS- Plus Brookfield Remoter was used for determination of rheological behavior of TFG and TI polysaccharides. It comprises of concentric cylinder, measuring cones and plates geometry. C-25 Din measuring system was used for the study. Temperature was regulated by using Peltier thermo regulator (Brookfield PTR-I) and obtained rheological data were recorded with the software RHEO 2000 version 2.8. All the rheological measurements were made in triplicate and the average of data were reported. Solution of different concentrations (1.0 to 4% w/v) of TFG and TI was prepared in deionised water, under slow stirring for 4 h using a magnetic stirrer (5MLHDX, Remi equipments Pvt. Ltd). Resulted solution was stored overnight for complete hydration. Flow behaviour of both the polysaccharide solution were examined under a range of shear rate (s^-1) from 10 to 100 in 100 sec followed by immediately decrease from 100 to 10 in 100 sec. Sample was placed in measuring gap between the rotating cone or plate and the stationary lower plate, allowed to equilibrate for 5-10 min. Flow behavior index (n) and consistency coefficient (k) were calculated from curve by fitting the power law model equation 7.

τ = kγⁿ (7)

Where τ is shear stress (Pa), γ is shear rate (s^-1), k is consistency coefficient (Pasⁿ) and n is flow behavior index (dimensionless).

2.2.6. Assessment of In vitro Cytotoxicity:

In order to performed cell toxicity of extracted polysaccharides, MTT assay was performed by using rat macrophage cells. The fresh cell lines were maintained in essential media supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL) in a humidified atmosphere at 37±0.5^oC of temperature. Different concentrations (ranging from 10 to 30 µg/ml) of aqueous solutions of extracted polysaccharides (TFG and TI) was prepared in sterile double distilled water. The cells were incubated for specific time period with different dilution of sample solutions of polysaccharide into 24-well plates in 100 µl of respective media. Plating density of cell were maintained 1x10⁵/well. After specific time period, sample solutions were removed from the cell and washed with phosphate buffer saline (pH 7.4). After that 200 µl/well (5mg/mL) of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) in phosphate buffer saline solution was added and incubated for 4 h. viability of cells were determined by the absorbance at 570 nm. Finally, percentage cell viability was calculated in respect of positive control by using following equation.

absorbance of test sample

% cell viability= --------------------------- x 100

absorbance of positive control (8)

3. RESULT AND DISCUSSION:

3.1. Organoleptic and Micrometric Properties:

Plant polysaccharides were extracted as per the method described in section 2.1. The percentage yield of TFG and TI were found to be 12.8 % w/w and 9.85% w/w respectively. Organoleptic and micrometric evaluations of both polysaccharides were performed and results are summarised in table 1. pH of both polysaccharides were investigated to check there acceptability with the biological mucosa. The study showed that both the extracted polysaccharides have pH within physiological range, hence they are non – irritating to the biological mucosa. Micrometric properties have significant importance in pharamaceutical industry. The result of micrometric evaluation of TFG and TI were represented in table 1. Bulk density of TFG and TI found 0.519±.004 and 0.497±0.02 respectively, which helps in selecting the proper size of a container, packaging material, mixing apparatus in the production of dosage form. Tapped density was determined to find powder density when air spaces and voids are removed which found 0.706±0.016 and 0.709±0.012 for TFG and TI respectively. Values of angle of repose of TFG and TI indicated that flow property of powder was good and excellent respectively. All micrometric parameters were found in the acceptable range indicated that both polysaccharide may be used as pharmaceutical excipients.

Table 1: Organoleptic and Micrometric Characteriztion of TFG and TI Hydrogel

	*Trigonella* **foenum graecum**	*Tamarindus* *indica*
Colour	Light Brown	Greenish
Odour	Characteristic	Odourless
Taste	Mucilaginous	Mucilaginous
pH	7.476±0.145	7.86±0.643
Bulk density (g/ml)	0.519±.004	0.497±0.02
Tapped density (g/ml)	0.706±0.016	0.709±0.012
*Angle of repose(θ)**	33.91±1.21	26.56±0.12
Carr’s index (%)	22.42±2.37	21.88±1.2

3.2. Surface Characteristics by SEM: -

To study the surface characteristics of extracted polysaccharides, SEM images was taken (Figure 1). The SEM of dry powder of TFG and TI exhibited rough surface with pores and crevices on it. Earlier, it has been reported that the drug release from the dosage form depends on surface characteristics of excipients. The surface roughness of the polymer is responsible for greater mucoadhesion and also for the retardation of drug release due to the entrapment of drug particles in the pores and crevices. Hence, it can be stated that both the mucilage can sustain the drug release because of their rough surface. From the SEM, it was also evident that the particle size of both the powders was not uniform and the size distribution was not within a narrow range. The powder contains larger to ultra fine particles. This might be the reason for the ‘heavy’ nature of the powders. The powders exhibit a ‘closet’ packing arrangement, in which, the smaller particles fill the voids between larger particles and reduce the bulkiness. This packing arrangement is indicated by the low total porosity values as well [11].

Figure 2: SEM images of Trigonella foenum graecum and Tamarindus indicia polysaccharides

3.3. Determination of Glucose:

The standard curve of glucose at different concentration is illustrated in figure 2. The Calibration curve was found to be linear with correlation coefficient of 0.997. From the value of slop and intercept, the concentration of glucose in test solution of polysaccharide was calculated. Mean value of total percentage glucose concentration in TGF and TI was calculated 23.5±4.72 and 31.9±6.32 respectively. The percentage relative standard deviation (% RSD) was found 0.61, which indicates that the used method was precise and accurate.

Figure 2: Standard curve of glucose at concentration range from 10 to 100 µg/ml.

3.4. Molecular Weight:

The experimental data for molecular weight determination of both extracted polysaccharide are provided in Table 2. Average molecular weight of TFG and TI was calculated 2.03 x 10⁶ kg/kg mol and 1.79 x 10⁶ kg/kg mol respectively by interpolating the line of graph between HC/τ and C, till zero concentration, as reciprocal value of intercept.

3.6. In vitro Cytotoxicity Study:

In order to access cytotoxicity of extracted polysaccharides (TFG and TI), MTT assay was performed. Obtained data of absorbance of test samples and positive control sample were incorporated in equation 8. Result of cell toxicity study of TFG at 10, 20 and 30 µg, demonstrated that the % cell viability compared to its positive control was 84.39, 89.39 and 83.94 respectively. Similarly, TI at same concentration possess 89.95, 80.59 and 87.58% cell viability compared to positive control. Cell viability study indicates that none of the dilution of any extracted polysaccharide possess cytotoxic effect on rat macrophage cells therefore, may be considered as safe excipients during delivery.

CONCLUSION:

The present study concludes that both plant polysaccharides (Trigonella foenum graecum) and (Tamarindus indica) can be considered as effective pharmaceutical excipients for drug delivery. physical state of polysaccharides such as micrometric properties, molecular weight and rheological properties was in accordance to the existing used polymers. Cell viability study also confirm that polysaccharide will be nontoxic during administration. However, further studies are required to established the complete profiles of these polysaccharides.

CONFLICT OF INTEREST:

Author declare no conflict of interest.

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Received on 28.11.2016 Modified on 11.12.2016

Research J. Pharm. and Tech. 2017; 10(1): 227-232.

DOI: 10.5958/0974-360X.2017.00048.8

Concentration (kg/m³)	Refractive index (n)	Turbidity (τ)	HC/τ	M_w,ave(kg/kg mol)
TFG polysaccharide
2.5	1.3384	101	1.74068x10^-6
5	1.3391	115	2.01394x10^-6
10	1.3403	126	2.39484x10^-6	2.03x10^-6
15	1.3484	158	2.48588x10^-6
20	1.3543	178	2.81628x10^-6
TI polysaccharide
2.5	1.283	79	2.34665 x10^-6
5	1.302	82	2.95541x10^-6
10	1.327	88	3.29349x10^-6	1.79 x 10⁶
15	1.232	91	3.49953x10^-6
20	1.346	98	4.00384x10^-6

Concentration (%w/v)	Flow behaviour index (n)	Consistency Coefficient (k)	Correlation coefficient (r²)	Flow behaviour
TFG polysaccharide
1	0.3846	1.1623	0.9397	Pseudoplastic
2	0.3192	1.2442	0.9763	Pseudoplastic
3	0.1835	1.6823	0.9815	Pseudoplastic
4	0.2548	1.7273	0.9852	Pseudoplastic
TI polysaccharide
1	0.3549	1.7746	0.9117	Pseudoplastic
2	0.3343	1.8353	0.9476	Pseudoplastic
3	0.2835	2.1371	0.9526	Pseudoplastic
4	0.2384	2.2739	0.9912	Pseudoplastic