ISOLATION OF D-TSP FROM TAMARIND KERNEL AND ITS ...

RESEARCH ARTICLE

Kamrun et.al / IJIPSR / 4 (10), 2016, 1029-1044

Department of Pharmacy

ISSN (online) 2347-2154 DOI: 10.21276/IJIPSR.2016.04.10.491

International Journal of Innovative Pharmaceutical Sciences and Research www.ijipsr.com ISOLATION OF D-TSP FROM TAMARIND KERNEL AND ITS EVALUATION AS AN AFLATOXIN-FREE NATURAL PHARMACEUTICAL EXCIPIENT 1

1

Mst. Kamrun Nahar*, 1Ismet Ara Jahan, 1Husna Parvin Nur, 2Md. Matiur Rahim, 1 Nurzaman Ara Ahmed, 1Satyajit Roy Rony

Bangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories, Dhaka. BCSIR, Dhaka-1205, BANGLADESH 2 Institute of Food Science & Technology (IFST), BCSIR, Dhaka-1205, BANGLADESH Abstract The purpose of this work was to enhance the source of natural polysaccharide which could be used as pharmaceutical excipient instead of synthetic ones. In this paper, polysaccharide was isolated from defatted tamarind kernel powder (particle size > 63 - 177 μ). The protein content was smaller (7.02 ± 0.2 %) compared to the previous study (12.6% - 15.2%) and it was free from the most concerning poisonous and cancer causing chemicals such as aflatoxins (AfG1, AfG2, AfB1, AfB2) contamination. This makes it safe and more applicable to be used in pharmaceuticals. Other safety measurement such as microbial testing was tested, which did not support of microbial growth and pathogenic organism. The defatted tamarind seed polysaccharide was primarily identified using Ruthenium red test and also by spectroscopical and structural investigations such as FTIR, X-ray diffraction, and SEM. Further phytochemical screening, physicochemical analyses (PH- 6.54 ± 05, viscosity- 4000 to 5000 cP, bulk & tapped density were 0.51 ± 0.02 & 0.72 ± 0.01 g/cc respectively, compressibility index- 2, swelling index- 1700 ± 0.5%, water retention time/g- 20.00 ± 1.5%), and minor components analyses (organic & inorganic components; Pb, Cd were below the detection limit) were conducted. The result of all carried out analyses support that it could be an excellent natural pharmaceutical excipient.

Keywords: Tamarind seed, excipients, aflatoxin, microbiological test, PH tolerance. Corresponding Author: Mst. Kamrun Nahar Senior Scientific Officer Chemical Research Division, BCSIR Laboratories, Dhaka. BANGLADESH Email: [email protected] Phone: +8801717134805 Available online: www.ijipsr.com

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INTRODUCTION Excipients are vital part of medicinal compound, which may also be a major portion of the medicinal products. They are inert molecules that play a very important role in the designing of a dosage form [1]. Natural pharmaceutical excipients nowadays take over the synthetic ones because they are economical, readily available, non-toxic, and capable of chemical modifications, potentially biodegradable, and also biocompatible with few exceptions [2,3]. Natural polysaccharides which are biodegradable and hydrophilic in nature are extensively used for the development of solid oral dosage forms [4]. Research had been conducted on gum copal and gum damar- based sustained drug delivery matrix [5], on xanthan gum-based sustained release matrix tablet of diclofenac sodium [6] and tamarind seed polysaccharide as an excipient for sustaining release of verapamil hydrochloride [7]. At present several pharmaceutical excipients of plant origin like starch, agar, alginates, carrageenan, guar gum, xanthan gum gelatin, pectin, acacia, tragacan and cellulose are being used [8]. Defatted tamarind seed polysaccharide (D-TSP) is a xyloglucan extracted from defatted tamarind kernel powder obtained from seeds of Tamarind tree (Tamarindus indica), which is a large evergreen tree belonging to the family Fabaceae. It possesses properties such as high viscosity, broad pH tolerance and adhesivity [9] which led its application as stabilizer, thickener, gelling agent and binder in food and pharmaceutical industries. Moreover, recently identified properties are non-carcinogenicity [10], mucoadhesivity, biocompatibility [11], high drug holding capacity [12] and high thermal stability [13]. For this reason it finds its application as excipient in hydrophilic drug delivery system [11]. Tamarind seed polysaccharide has an average molecular weight of 52,350 Dalton and it is also called galactoxyloglucan [12]. The objectives of this work were as follows. Firstly to isolate fat free tamarind seed polysaccharide, to evaluate the polysaccharide as a safe natural pharmaceutical excipient and this could be used instead of synthetic ones and to examine whether this polysaccharide was free from aflatoxin contamination.

MATERIALS AND METHODS PLANT MATERIAL & CHEMICAL REAGENTS Seeds of Tamarind (Tamarindus indica) had been collected from Kaoran Bazar, Dhaka, Bangladesh. The solvent ethanol and other chemicals used were of analytical grade. Available online: www.ijipsr.com

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D-TSP ISOLATION METHOD D-TSP was isolated by aqueous solution and non-aqueous precipitation method. Dried tamarind seed was heated with preheated sand for removing outer shell by mild crushing. Perfectly clean kernel was powdered (TKP) and free fat was removed using n-hexane by soxhlet method. This Defatted Tamarind Kernel Powder (D-TKP) (up to 100 micron) was then taken into different fractions by air classifying. The first fine fraction (< 10 μ, 15 – 30 %) was discarded but the second coarse fraction (>63 - 177 μ) was taken for isolating defatted TSP. The desired fraction (20 g) was mixed with cold distilled water (200 ml) to prepare slurry. The obtained slurry was then poured into 800 ml of hot distilled water and boiled on a water bath at 90 to 100 ⁰C with stirring for 1 to 2 hours. It was then kept aside for overnight at room temperature for mart to settle down and also for the release of mucilage. After this the solution was filtered with silk cloth to remove mart. The filtrate was then condensed by heating. The filtrate was again cooled at room temperature and equal amount of ethanol was added to result in the precipitation of polysaccharide. The precipitation process was carried out at room temperature. The precipitate was then dried at 600C for 5-6 hours. The dried sample was powdered and sieved with mesh no. 80 (177 μ). The yield was 50 – 60 % on the dry basis. IDENTIFICATION TEST Ruthenium red test was performed to confirm the nature of mucilage obtained [14]. PHYTOCHEMICAL SCREENING Phytochemical screening was conducted to confirm the purity of the product such as test for Carbohydrates (Molich‟s test + Iodine test), test for Tannins (Ferric chloride test), test for flavonoids (Shinoda test), test for chlorides (silver nitrate test) and test for sulphates (barium chloride test). PHYSICOCHEMICAL TESTS Moisture content and total ash content were determined using AOAC Official Method (2005), and pH (1% w/v solution) was determined by Jenway (bench type) PH meter at 25oC. Viscosity was measured by Plate and Cone method using the viscometer; Haake, VT-550, Germany at 100 rpm, 25°C in PP & PDC, BCSIR. Sample of 1% w/v solution was taken with the help of vortex meter for dissolving sample in distilled water. Bulk Density, Tapped Density, Compressibility Index and Hausner Ratio were measured by World Health Organization, Document (2012) in CRD, BCSIR Labs, Dhaka. The swelling index of D-TSP was determined using a method described in “Quality Available online: www.ijipsr.com

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control methods for medicinal plant materials,” (WHO, 1998). Water retention capacity of defatted TSP was determined using the modified method which was reported by Gautami & Ramesh [15]. The crude protein was determined by Automated Kjeldahl method of AOAC Official Method 976.05 (2005) in IFST, BCSIR and the process was carried out in macro Kjeldahl flasks with side arms, which were rotated at 3 min intervals through each successive step. Fiber (crude) was determined using Fritted Glass Crucible Method of AOAC Official Method 978.10, (2005).The residual fat was determined by Hexanes Extraction Method of AOAC Official Method 2003.06 (2005). The minerals were determined using Atomic Absorption Spectrophotometric Method under AOAC Official Method 968.08. The minerals Ca, Mg, Fe, Cd, pb, Ni, and Cr were measured using a GBC 908 Atomic Absorption spectrophotometer. Phosphorous (P) was determined calorimetrically by the ammonium molybdate method using a Shimadzu Ultraviolet Visible spectrophotometer 1601 PC. K and Na were analyzed using a Corning 410 flame photometer. SAFETY MEASUREMENT OF D-TSP The crystalline aflatoxins were dissolved in benzene acetonitrile (98:2) to obtain a concentration of 0.5 μg/ml aflatoxin B1 and G1, and 0.25 μg/ml for B2 and G2. Appropriate aliquots were taken to give specific concentration of the individual aflatoxins. The AOAC method (970: 40 1990) was followed for the extraction and purification of aflatoxins from extracted sample in brief: 250ml of acetone-water (1:4) was added to the finely grounded sample (50 g) and shaken mechanically at moderate speed for 30 min, then filtered through a whatman no. 1 filter paper in a quickfit conical flask. Ten milliliters of filtrate was transferred into a 250 ml measuring cylinder, where 1 ml of lead acetate and 10 ml of methanol were added using dispensers and then distilled water was added to make a volume upto 150 ml.The SPE (Solid Phase Extraction) clean-up procedure was performed with a Phenyl Bond Elute Column. The column was washed with 10 ml distilled water and then dried. A 7 ml glass vial (suitably labeled) was placed in the vacuum apparatus to serve as a collection vessel. A sodium sulphate column was fit below the phenyl column to dry the final extract. Alatoxins (if any) were eluted from the SPE cartridge with 4 ml elution solution. The vial was dried at 40-50 ºC under a stream of dry nitrogen in the sample concentrator (It did not allow going to total dryness). The sample was reconstituted with 1 ml mobile phase and vortex briefly, and filtered through syringe filter into an HPLC auto sampler vial. The HPLC equipment was an Agilent reverse phase HPLC system (series 1100, Germany) with FLD (florescence detector). The mobile phase was 630 ml Available online: www.ijipsr.com October Issue 1032

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water, 220ml methanol, 150 ml acetonitrile, 120 μL of concentrated nitric acid and 100 mg potassium bromide in isocratic mode with 1 ml flow rate. The aflatoxin concentrations were determined and quantified by the retention time and peak areas, respectively. Total run time was 15 min and injection volume was 20 μL. Column oven temperature was 30°C and excitation wavelength was 365 nm, and emission wavelength was 464 nm. The method was validated as per Food & Feed Safety of European commission decision, 2002. Enumeration of Total Aerobic Plate Count [16], Total Fungus Count [17], Escherichia coli [18], Salmonella [19], and Staphylococcus aureus [20] were carried out by following Bacteriological Analytical Manual. Total Plate Count was enumerated by Pour Plate Method on melted nutrient agar/ plate count agar. Enumeration of fungus was carried out by spread plate technique on potato dextrose agar (PDA) media. Enumeration of Pseudomonas aeruginosa and Staphylococcus aureus were carried out by selective method where Baired- Parker ager medium and cetrimide agar plates were used for the detection of Staphylococcus aureus and Pseudomonas aeruginosa, respectively. Escherichia coli count was carried out by using three-tube most probable number (MPN) method. Lactose broth and eosin methylene blue (EMB) agar were used for E. coli. Salmonella count was carried out by using Enrichment method where Lactose broth, selenite broth and bismuth sulphite agar were used. Total Plate Count of Escherichia coli, Staphylococcus aureus and fungus were enumerated on plate count agar. Incubation temperature and time were 37oC and 24 hours, respectively. Suspected pathogens were biochemically confirmed. SPECTROSCOPIC AND STRUCTURAL INVESTIGATION OF D-TSP A spectrum of D-TSP was obtained by using Fourier Transform Infrared Spectroscopy (FTIR) by ATR (Attenuated total reflection) method in BCSIR Laboratory, Dhaka. The sample was scanned from 4000 to 400 cm–1 in a BRUKER spectrophotometer, where the typical material for ATR crystal was germanium. Diffraction pattern of powdered sample was recorded with an X-ray diffractometer (BRUKER, D8 Advance, and Germany) in the laboratory PP & PDC, BCSIR. X-ray diffraction was performed at room temperature (30oC) with a diffractometer; target, Cu Kα (λ = 1.5406 Å), filter, Ni; Voltage, 40 kV; current 40 mA; time constant 10mm/s; scanning rate 2o/min; measured from 10-35o at full scale 200. The SEM images of TKP, D-TKP & D-TSP were taken by SEM (Hitachi-2600SN, Japan) without coating and the images were taken with 10.0 KV voltages at 12.5 mm x 800 and 12.4 mm x 250 magnifications respectively in Industrial Physics, BCSIR Laboratory, Dhaka. Available online: www.ijipsr.com October Issue

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RESULTS AND DISCUSSION RESULT AND DISCUSSION ON PRECAUTION TAKEN FOR THE PURIFICATION OF D-TSP WHILE ISOLATION For isolation of D-TSP, D-TKP was taken with particle size > 60 - 177 micron, as fine fraction having an increased protein content and a decreased polysaccharide content, and a coarse fraction having reduced protein content and increased polysaccharide content [21]. The precipitation process was carried out at room temperature as organic solvents denature proteins at room temperature. RESULTS AND DISCUSSION OF IDENTIFICATION, PHYTOCHEMICAL AND PHYSOCOCHEMICAL INVESTIGATION The isolated polysaccharide was stained with ruthenium red followed by irrigation with lead acetate and observed the development of pink color which showed the presence of D-TSP (mucilage). The phytochemical investigation also showed the presence of carbohydrates, and absence of tannins and flavonoids (Table-1), which indicates its purity. The natural polymers always have unique properties which make them different from the synthetic polymers, and D-TSP is also not exception from this, as it shows a wide range of properties making it an effective polymer in the field of pharmaceutical industries. It was dissolved completely in hot water above 85 °C, yielding a highly viscous colloidal solution or a mucilaginous gel which was relevant to the work of Poommarinvarakul et al. 2010 [22]. It possessed various properties (Table-2) such as high viscosity, broad pH tolerance and nonhygroscopic nature. This has led to its application as stabilizer, thickener, gelling agent and binder in food and pharmaceutical industries. It showed swelling index and high thermal stability, making it a suitable excipient for drug delivery system which was relevant to the work of Avachat et al. 2011 [23]. Another investigation on TSP showed that it possessed important properties such as; non-carcinogenicity, mucoadhesivity, biocompatibility, high drug holding capacity and high thermal stability. This has led to its application as excipient in hydrophilic drug delivery system [10, 11]. Mucoadhesive polymer exhibits its adhesiveness only when it is in contact with water or gastro intestinal solution and D-TSP showed this property. It was also found to be a potential emulsifier, nontoxic and non-irritant with haemostatic activity [23, 24, 12]. Apart from this, it was also an excellent viscosity enhancer showing mucomimetic, mucoadhesive and bioadhesive activities [25]. Available online: www.ijipsr.com

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The powder and the solution properties (Table No: 2) of D-TSP, such as density, flow, compressibility, melting point, moisture content, water retention, swelling index, pH, and surface tension were evidently proved to be satisfactory for its applications in various fields, especially for pharmaceutical formulation development which has been relevant to the study of Phani [24]. Another investigation on TSP showed that it played the role as stabilizer, thickener, binder, release retardant, modifier, suspending agent, viscosity enhancer, emulsifying agent, and as a carrier for novel drug delivery systems for oral, buccal, colon, ocular systems, nanofabrication, wound dressing, food, cosmetics, confectionary, bakery, etc [26]. The widespread use of powders in the pharmaceutical industry has generated a variety of methods (angle of repose, compressibility index or Hausner ratio etc) for characterizing powder flow. In a free flowing powder, the bulk and tapped densities will be closer in value, and in this study DTSP was found to possess these characters. D-TSP possessed high viscosity (4000- 5000 cP of 1% w/v), broad pH value (6.54 ± 05 of 1% w/v), low moisture content (7.65%), high swelling index (1700 ± 0.5 %), and decomposition temperature (240- 260º C). The obtained Compressibility Index was 2 (Compressibility Index % ≤ 10, excellent flow property); the Hausner Ratio was 1.41 (Flow character excellent when it is 1.00-1.11); angle of repose: 31.89 (Flow property Good); (U.S. Pharmacopeia, 1965, pp. 163-168).The physicochemical properties of D-TSP are shown in Table No: 2. Table 1: Phytochemical screening of D-TSP Tests

Observation

Test for Carbohydrates (Molich‟s test + Iodine test )

+

Test for Tannins ( Ferric chloride test )

-

Test for mucilage ( Ruthenium red test)

+

Test for flavonoids ( Shinoda test)

-

Test for chlorides( silver nitrate test)

-

Test for sulphates( barium chloride test)

-

„+‟ indicates presence, „-‟ indicates absence Available online: www.ijipsr.com

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Table 2: Physicochemical properties of D-TSP Parameters Appearance Odor Color Solubility Taste Moisture (%) Total ash content (%) pH ( 1% w/v solution); (Jenway, bench type) at 25oC Viscosity (1% w/v solution) at25oC (cP) Decomposition temperature (0C) Bulk density (80 mesh) (g/cc) Tapped density (80 mesh) ( g/cc) Compressibility index (80 mesh) (%) Angle of repose (80 mesh) (⁰) Swelling index in water (%) Water retention /g (%)

Results Granular powder Odorless Cream color Soluble in hot water Tasteless 7.05 ± 0.02/ 0.59 ± 0.01 6.54 ± 0.05 4000 – 5000 240 – 2600C 0.51 ± 0.02 0.72 ± 0.01 2.0 ± 0.01 31.89 ± 0.01 1700 ± 0.5 20.00 ± 1.5

Values are given in means of triplicate measurements. RESULTS AND DISCUSSION ON COMPOSITION OF MINOR COMPONENTS Crude protein found in D-TSP was 7.02-7.44% (Table No: 3), but the finding of Reddy (2011) on polysaccharide from tamarind kernel, the value was (12.6- 15.2%) which was greater than the present findings [27]. Residual fat and crude fiber were not observed. The purity of this isolated D-TSP was also proven by the results of its composition of minor components. Contamination of medicinal plant materials with heavy metals can be attributed to many causes including environmental pollution and traces of pesticides. Heavy metals like Cd (0.07 ppm) and Cr (1.22 ppm) were found in negligible amount, and Pb & Ni are below the detection limit. On the other hand, the nutrient inorganic components like Na, K, Ca, Fe, P (as PO4), and Mg were in acceptable limit as shown in Table No: 4. From the analysis it has been shown that the heavy metals were in acceptable limit established by European Medicines Agency (EMEA, 2008), shown in Table No: 4. Table 3: Organic components analysis in D-TSP Compound Present Crude protein

Percentage 7.02 ± 0.2

Crude fiber

-

Residual fat

-

Non-fiber carbohydrate

84.92 ± 0.2

Values are given in means of triplicate measurements, „-‟ indicates absence Available online: www.ijipsr.com October Issue

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Table 4: Inorganic components analysis in D-TSP and comparison with Reference Limit (“European Medicines Agency (Pre-authorisation Evaluation of Medicines for Human Use),” 2007) Concentration Limits for Individual Metal Catalysts and Metal Reagents (Reference Limit) Oral Exposure Parenteral Exposure Concentration Concentration (µg/day) (µg/day) (ppm) (ppm) (5.75 X 105 to ---3.450 X 106)a

Parameters

Results (ppm)

Na

19.31

K

678.6

(31 X 105 )b

--

--

--

Ca

279.5

(7 X 105, )c

--

--

--

Mg

166.0057

--

--

--

P (PO4)

874

(15 X 104 5 X 105)d --

--

--

--

Fe

50.33

13000

1300

1300

130

Cd

0.07

--

--

--

--

Pb

BDL

100

10

10

1

Ni

BDL

300

30

30

3

Cr

1.22

300

30

30

3

BDL= Below Detection Limit. a

indicates that, the acceptable range of sodium intake for adults according to European

Food Safety Authority, 2006. b

indicates the average dietary intake of potassium according to European Food Safety

Authority, 2009. c

indicates that acceptable range of calcium intake for adults according to European Food

Safety Authority, 2009. d

indicates that acceptable range of magnesium intake according to European Food Safety

Authority, 2009. RESULT AND DISCUSSION ON SAFETY MEASUREMENT D-TSP is recommended for pharmaceutical excipient as it did not support for the growth of microbiological and pathogenic organisms. (Table No: 5) and the result was supported by Sravani et al. 2012 [28] where total microbial count determined by streak plate method showed negligible amount of microbes at the end of 7th day of the study (46 CFU/g).

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Aflatoxins are metabolic products of the molds Aspergillus flavus and Aspergillus parasiticus, may occur in stored agricultural crops (such as peanuts and other nut crops) when growth conditions and genetic requirements are available [29]. The common aflatoxins are B1, B2, G1, and G2 where B1 is the most toxic followed by G1; the toxicities of B2 and G2 are relatively weak. The International Agency for Research on Cancer (IARC) concluded that there was sufficient evidence in humans for the carcinogenicity of naturally occurring aflatoxins (IARC 1993, 2002) (Report on Carcinogens, Thirteenth Edition; Aflatoxins CAS No. 1402-68-2). Table-6 shows that, D-TSP was free from aflatoxins (B1, B2, G1, and G2) contamination. So it could be an excellent excipient in pharmaceutical dosage when compared to other natural excipients such as maize and starch. An investigation by Khatoon (2012) on aflatoxins contamination in maize shown the values were: B1 (27.69%), B2 (18.46%), G1 (1.3%) and G2 (