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Structural, functional and pH sensitive release characteristics of water-soluble polysaccharide from the seeds of Albizia lebbeck L.
Accepted Manuscript Title: Structural, functional and pH sensitive release characteristics of water-soluble polysaccharide from the seeds of Albizia lebbeck L. Authors: Chekuri Ashok Kumar Varma, K. Jayaram Kumar PII: DOI: Reference:
S0144-8617(17)30884-6 http://dx.doi.org/doi:10.1016/j.carbpol.2017.08.017 CARP 12624
To appear in: Received date: Revised date: Accepted date:
6-7-2017 25-7-2017 3-8-2017
Please cite this article as: Kumar Varma, Chekuri Ashok., & Jayaram Kumar, K., Structural, functional and pH sensitive release characteristics of water-soluble polysaccharide from the seeds of Albizia lebbeck L.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.08.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural, functional and pH sensitive release characteristics of water-soluble polysaccharide from the seeds of Albizia lebbeck L. Chekuri Ashok Kumar Varma1, K. Jayaram Kumar1* 1
Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra,
Ranchi-835215, Jharkhand, India. *Corresponding author. Tel.: +91 06512276247; fax: +91 06512275290. E-mail addresses: [email protected] (K. Jayaram Kumar)
Highlights
Mainly comprises of glucose, rhamnose, galactose, xylose and mannose.
High swelling, water holding and foaming capacity were observed.
ALPS was found to be a non-ionic polysaccharide.
ALPS exhibited pH sensitive drug release.
Abstract Plant polysaccharides, generally regarded as safe (GRAS), are gaining importance as excipients in drug delivery. Therefore, the current paper presents the studies on structural, functional and drug release study of water soluble polysaccharide (ALPS) from seeds of Albizia lebbeck L. High swelling, water holding capacity, foam stability and lower moisture content suggests its use as additive in food preparations. The apparent molecular weight of polysaccharide was found to be 1.98 × 102 kDa. Monosaccharide composition analysis indicated that ALPS consists of mannose (4.06%), rhamnose (22.79%), glucose (38.9%), galactose (17.84%) and xylose (16.42%). Micromeritic properties revealed that the polysaccharide possess potential for pharmaceutical applications. From the surface charge analysis, ALPS was found to be non-ionic polysaccharide. Morphological study reveals the polysaccharide with irregular particle shape and rough surface. Fourier transformed infrared spectroscopy (FTIR) study confirms the carbohydrate nature of polysaccharide. From the thermogravimetric analysis (TGA) data, the second mass loss (243340oC) attributed to polysaccharide degradation. The drug release profile reveals the use of polysaccharide for the preparation of pH sensitive pharmaceutical dosage forms. Key words: water soluble polysaccharide, FTIR, pH sensitive.
1. Introduction Polysaccharides, one of the major class of biopolymers which are widely used in almost all sectors. As these polysaccharides are fermented by the colonic microflora, they falls into the category of GRAS (Kosaraju, 2005). Even though, there are large number of polysaccharides available in market but currently an extensive research is going on to explore the new sources of polysaccharides. This is due to huge demand of polysaccharides with desired characteristics in the market. These polysaccharides has a tremendous use in pharmaceutical sector such as binder, disintegrant, thickening and stabilizing agent (Kulkarni, Sinha, & Jayaram Kumar, 2013). These are also having tremendous role in targeting and controlled release of drugs. Water soluble polysaccharides, which are more preferable in the pharmaceutical sector, as they are easy to handle with regard to its high solubility and hydrophilic nature. Due to this peculiar characteristics of water soluble polysaccharide, further physical or chemical modification process makes easy to obtain the desired characteristics of polymer (Munir, Shahid, Anjum, & Mudgil, 2016). From the point of drug delivery, few drugs requires targeting to particular site either for the absorption from gastrointestinal (GI) system or for site specific diseases. The pH-dependent systems are generally accepted to fulfill this requirement, as the pH of GI tract increases from stomach (pH 2-3) to colon (7-8) (Khan, Stedul, & Kurjaković, 2000). Taking this advantage, natural pH sensitive polysaccharides without any tailored made process are need to be explored. Albizia lebbeck L., the Indian siris, flea tree or fry wood, belonging to the family Leguminosae, Mimosoideae (Al-Massarani et al., 2016; Shaikh, Gadge, Shinde, Padul, & Kachole, 2014). It is a perennial tree, which is widely distributed in India and adopted to other tropical and sub-tropical regions (Al-Massarani et al., 2016; Lam & Ng, 2011). It produces a seed pods with a length of 1530 cm and contains 6-12 seeds (Bobby, Wesely, & Johnson, 2012; Missanjo, Maya, Kapira, Banda,
& Kamanga-Thole, 2013). The seeds are consumed for the treatment of piles, diarrhea and scrofulous swelling, whereas the seed oil is used for the treatment of leprosy (Bobby, Wesely, & Johnson, 2012; Gupta, Kachhawa, & Chaudhary, 2006). These pods comprises of cellulose (36.4%), hemicellulose (8.9%), lignin (3.6%), and volatile matter (83.1%) (Ahmed & Theydan, 2014). As the seeds are rich in polysaccharides, the current study focuses on exploration of water soluble polysaccharide from Albizia lebbeck L. seeds and to characterize further in terms of structural, functional and morphological characteristics. Finally, evaluation of the isolated polysaccharide for tablet preparation and to study its drug release profile. 2. Materials and methods 2.1. Materials Albizia lebbeck L. pods were collected from B.I.T. Mesra, Ranchi and authenticated at botanical survey of India, Kolkata with specimen no. AL-01. 3- Methyl-1-phenyl-2-pyrazoline-5-one (PMP) was purchased from Sigma Aldrich. Sugar standards (Arabinose, Glucose, galactose, xylose, fucose, rhamnose and mannose) were purchased from Hi-Media. All other reagents used were of analytical grade. 2.2. Isolation and purification of Polysaccharide The pods of Albizia lebbeck L. were dehusked to obtain the seeds, which were then washed with distilled water. The seeds were then steeped in distilled water for 24 h. The imbibed seeds were then boiled for 8 h and the obtained slurry was kept at 4-8 oC for 12 h, followed by centrifugation at 5000 RPM. The resulted supernatant fluid was then precipitated in 95 % ethanol. Further, the suspension was centrifuged and then dialyzed with distilled water for 24 h. Finally, the dialyzed suspension was lyophilized and further purified by using gel permeation chromatography containing Sepharose-6B column (90cm x 2.1cm) using deionized water as eluent with a flow rate
of 0.4 ml/sec (Nandan, Sarkar, Bhanja, Sikdar, & Islam, 2011). 4 ml of eluent was collected in each test tubes and measured spectrophotometrically by using phenol-sulphuric acid method at 245 nm (York, Darvill, McNeil, Stevenson, & Albersheim, 1985). A plot of number of test tubes versus absorbance was shown in Fig. 1. The homogeneous fraction was collected with a test number ranging from 16-21 and lyophilized to yield water soluble polysaccharide (ALPS). Molecular weight determination Gel-chromatographic technique was used to determine the molecular weight of polysaccharide by using standard dextrans (T-40, T-60, T-70, T-200 & T-250) (Nandan, Sarkar, Bhanja, Sikdar, & Islam, 2011). Initially, the standard dextrans were passed through Sepharose-6B column and the elution volumes were plotted against their respective molecular weights. The apparent molecular weight of ALPS was determined by comparing the elution volume with the standard dextrans.
2.3. Monosaccharide composition analysis Monosaccharide composition was determined by means of reverse phase high pressure liquid chromatography using pre-column derivatization process (Sun et al., 2009). To 1 ml of 0.5mg/ml polysaccharide, 1 ml of 4M trifluoroacetic acid (TFA) was added and heated on a water bath at 120 oC for 2 h. Further, the excess of TFA was removed by co-distillation with methanol. To the dry hydrolysate, 1 ml of NaOH and 1ml of 0.5 M methanolic PMP was added. Then the mixture was incubated at 70 oC for 1 h and then cooled, followed by addition of 1 ml of 0.3M HCl solution. Further, the mixture was centrifuged for 5 min and the supernatant containing labelled carbohydrates were filtered through 0.22 µm nylon filter. Finally, the resulting mixture was injected into C18 - SPHERISORB column (Waters, 4.6 mm x 150 mm) connected with a PDA detector (245 nm). 0.1 M phosphate buffer (pH-6.7) -acetonitrile (83:17) was used as a mobile
phase at a flow rate of 1ml/min. The identification and quantitative determination of sugars was determined by comparison with the standard sugars (D-glucose, L-arabinose, D-xylose, L-fucose, D-galactose, L-rhamnose and D-mannose). The molar ratio of monosaccharide was calculated from the peak area. 2.4. Determination of functional properties Functional properties of isolated polysaccharide-ALPS was done by determining the moisture content, pH, swelling and solubility power, water holding capacity and fat binding capacity. The pH of 1 % ALPS solution was determined by using digital pH meter. 2.4.1. Water-holding capacity (WHC) WHC was determined according to the method described by Deepika, Jayaram Kumar and Anima (2013) with slight modifications. A suspension of polysaccharide (1 g) in distilled water was prepared and kept under magnetic stirring for 1 h. The resulted suspension was centrifuged at 3000 rpm. The settled polysaccharide was weighed (WS) after decanting the supernatant. The WHC was calculated by the following equation; 𝑊𝑆 𝑊𝐻𝐶 = ( ) × 100 𝑊 Where W is the weight of dried polysaccharide. 2.4.2. Fat binding capacity (FBC) The fat-binding capacity was determined by the method described by Abdul-Hamid and Luan (2000). The polysaccharide suspension was prepared by suspending 0.5 g of polysaccharide in 10 ml of soybean oil and agitated for 30 min at a time interval of every 5 min. The suspension was then centrifuged at 3000 rpm for a duration of 10 min. The supernatant free oil was removed and decanted for 30 min on a filter paper. The fat binding capacity was computed by the following equation:
𝐹𝑎𝑡 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 (𝑔 𝑜𝑖𝑙/𝑔) =
𝑊𝑟 − 𝑊 𝑊
Where Wr is the weight of residual polysaccharide; W is the weight of dried polysaccharide. 2.4.3. Swelling and solubility power The swelling and solubility power of polysaccharide was determined by the method described by Kumar, Varma, and Panpalia (2014). The polysaccharide suspension was subjected to heating on a water bath at 30 °C, 60 °C, 90 °C for 30 min with occasional stirring to prevent lump formation. The suspension was allowed to cool and centrifuged at 3000 rpm for 15 min. The supernatant was transferred into a pre-weighed petri-dishes and dried for 2 h at 130 °C. The residue obtained after drying the supernatant was weighed and it represents the soluble portion of polysaccharide. The swelling power and solubility were calculated as: 𝑊𝑠𝑢 ⁄𝑊 ) × 100 𝑖
% 𝑆𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 = (
𝑊𝑠𝑠 % 𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 = ( ) × 100 𝑊𝑖 × (100 − % 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦) Wsu is the weight of soluble residue in supernatant; Wi is the weight of starch on dry basis; Wss is the weight of wet starch (g). 2.4.4. Foaming properties Foaming properties of ALPS was evaluated by determining the foam capacity (FC) and foam stability (FS) according to the method followed by Shahidi, Han and Synowiecki (1995). Different concentration of ALPS (1%, 2%, 3%, 4% and 5 % w/v) were prepared and homogenized using IKA homogenizer (Germany) for 2 min. After the homogenization process, the whipped samples were transferred into a graduated measuring cylinder. The total volume was noted before (Vo) and
after (VT) whipping. Further, the volume (Vt) was noted at 10, 20, 30 and 60 min. FC and FS was determined by the following equation: 𝐹𝐶 (%) = (
𝑉𝑇 − 𝑉𝑂 ) × 100 𝑉𝑜
𝑉𝑡 − 𝑉𝑂 𝐹𝑆 (%) = ( ) × 100 𝑉𝑜 2.5.Micromeritic properties Micromeritic properties of ALPS was determined by performing bulk and tapped density, angle of repose, Hausner ratio and Carr’s index. These properties were determined by the methods described by Varma, Panpalia and Jayaram (2014). 2.6. Morphological characterization Morphology of ALPS was determined by scanning electron microscopy (JEOL, Japan, JSM6390LV). For the analysis, the powder sample was scattered on a double sided tape which is adhered to the aluminum stub and then coated with platinum to make sample conductive. The image was captured at an accelerating voltage of 10 Kv. 2.7.IR analysis IR spectra of ALPS was obtained by using Fourier transform infra-red spectroscopy (FTIR-8400 S, Shimadzu, Japan). The sample was blended with Kbr and pressed into pellets, operated in the frequency range of 4000-400 cm-1. 2.8. Thermogravimetric analysis
Thermogravimetric analysis was performed by using Thermogravimetric analyzer (DTG-60, Shimadzu, Japan. The sample was subjected to heating (10 oC/min) in the temperature range of 30-600 oC under nitrogen environment. 2.9. Determination of surface charge Surface charge of a polysaccharide was determined by means of a zeta potential measurement, followed by the method described by Pang, Deeth and Bansal (2015). Stock solutions of ALPS was prepared in MilliQ water by magnetically stirring and heating at 80 oC for 30 min. Further, the solution was allowed to stir for 2 h at room temperature. The solution was made up to 0.1 % concentration by diluting with MilliQ water. Then the stock solutions were adjusted to different pH (2.3, 4.0, 4.3, 5.3, 6.3). Finally, the zeta potential of polysaccharide solutions were measured by using Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK). 2.10. Preparation of tablets and their evaluation The ALPS polysaccharide was used to prepare the tablets by means of wet granulation and diclofenac sodium was used as model drug. Lactose was used as diluent, magnesium stearate and talc were used as lubricants. The tablets with different concentrations of polysaccharide (5, 7.5 & 10%) were prepared and evaluated for physical tests like weight variation, hardness, friability, disintegration time, drug content and cumulative drug release. Drug release study of tablets were determined according to the method described by Varma, Panpalia and Jayaram (2014). 2.11.
Drug release Kinetics
Drug release kinetic study was performed in order explore the release behavior of drug from the tablets. The kinetic study was best explained by applying Hixson–Crowell, Higuchi’s model and Korsmeyer–Peppas equations. 2.12. Statistical analysis
All the values of analysis were reported on the averages of triplicate analysis and was expressed by means ± standard deviation. 3. Results and discussion 3.1. Physicochemical characterization of polysaccharide The percentage yield of polysaccharide was found to be 12.72 % ± 1.17 and the pH was found to be neutral. The moisture content of ALPS was found to be 10.97 %, suggesting for its use in dosage form formulations. Water holding capacity was found to be 835.65 ± 0.22 %, which is higher than that of Cymodocea nodusa sulfated polysaccharide (R. Ben et al., 2016). FBC of ALPS (4.69 ± 0.15 g oil/g sample) was also found to be significantly higher than that of chickpea flour (3.15 g oil/g sample) (Mokni Ghribi et al., 2015). The swelling and solubility power determines the controlled drug releasing behavior of polysaccharide. The swelling power and solubility power of ALPS was found to be 6.09 ± 0.19 % and 83.6 ± 0.15%, which is higher than that of polysaccharide isolated form Pithecellobium dulce seeds (Bagchi & Jayaram Kumar, 2016). Foam capacity and stability helps in determining the interfacial properties of surfactants used in the formulation containing two immiscible phases. The foaming properties of ALPS were measured at different concentration from 1 to 5% and the results were shown in Table 1. At 0 min, the foam capacity was found to be concentration dependent and found to increase up to 4% and became constant after this point. At 10 min, the foam stability reduced to a concentration range of 1-3% and further reduced for 1-4% at 20 min. The of 4 % concentration remained constant after 20 min At the same time, the foam stability of 5% remains constant throughout the study. This suggests that ALPS can be used for the formulations where there is a requirement to stabilize the interfacial forces among the liquid-gas (Mokni Ghribi et al., 2015). So, the ALPS can be used in
the food industry for the preparation of milkshakes, marshmallows, ice creams (Tan& Gan, 2016); in cosmetics such as liquid soap, shaving cream. Molecular weight and monosaccharide composition The apparent molecular weight of ALPS was determined from the elution curve obtained from sepharose 6B column in comparison with standard dextrans (T-40, T-60, T-70, T-200 & T-250) (Nandan, Sarkar, Bhanja, Sikdar, & Islam, 2011). From the elution curve of ALPS (Fig. 1), the polysaccharide was found in the 16-21 test tubes and from the calibration curve of standard dextrans, the apparent molecular weight was found to be 1.98 × 102 kDa. Monosaccharide composition analysis were performed by reverse phase chromatography and shown in Fig. 2. ALPS was found to contain Mannose (4.06%), rhamnose (22.79%), glucose (38.9%), galactose (17.84%) and xylose (16.42%) with a molar ratio of 1:5.6:9.5:4.3:4. Micromeritic properties Micromeritic properties of ALPS were mentioned in Table 2. The ALPS was found to have fair flow with a low Hausner ratio and Carr’s index. The angle of repose reveals passable flow property of ALPS. This suggest the use of ALPS in pharmaceutical sector, where there is requirement of material transfer through feeding devices. 3.2.FTIR FTIR spectrum of ALPS was presented in Fig. 3A. A broad band around 3200-3400 cm-1 represents the O-H stretching and a peak at 1650 cm-1 represents the C-O stretching (Chien, Yen, Tseng, & Mau, 2015). The peaks between 1000-2000 cm-1 represents the pyranose ring of sugar molecule. A peak at 840 and 890 cm-1 may be attributed to α- and β glycosidic linkages. The above FTIR data reveals the characteristics of a typical polysaccharide.
3.3.Thermogravimetric analysis Thermogravimetric analysis of ALPS (Shown in Fig.3B.) was performed to determine the stages of decomposition of polysaccharide with respect to increase in temperature. The ALPS showed three stages of weight loss, where the first stage involves the loss of bound moisture from the polysaccharide. The second mass loss (243-340°C) involves the degradation of polymer which corresponds to the depolymerisation of the polysaccharide structure (Liu et al., 2016). Finally, the third mass loss is related to the oxidation of organic matter. 3.4.Morphology of isolated polysaccharide Morphology of ALPS was studied by using scanning electron microscope and presented in Fig. 4. ALPS was found to have irregular particles with irregular particle shape and rough surface (Munir, Shahid, Anjum, & Mudgil, 2016). This characteristic surface implies the passable flow property of polysaccharide.
3.5.Surface charge The Zeta potential of ALPS was measured at different pH (2.3, 4.0, 4.3, 5.3, 6.3). At all these pH, ALPS showed a zeta potential around zero or slightly negative values lesser than -35mV. This results confirms that ALPS is a non-ionic polysaccharide (Pang, Deeth, & Bansal, 2015).
3.6.Tablet evaluation properties Tablets prepared by using ALPS were evaluated for their physical properties and were tabulated in Table 3. In order to meet the desired tablet characteristics, a series of tablet evaluation tests has been mentioned in USP (USP, 2008). The friability is one of the physical test of tablets, where it
determines the property of tablet to withstand the physical forces that are faced during packaging and transportation. The friability of all the tablets was found to be less than 1 % and hence meets the desired criteria. The weight uniformity and drug content of all the tablets meets the specifications mentioned in USP (USP, 2008). 3.7.In-vitro drug release study Drug release profile of tablets prepared with different concentration of ALPS was mentioned in Fig. 5. From the drug release study, it was found that with increase in the concentration of ALPS there is an increased release. The reason behind this might be due to high soluble content with increase in the concentration of ALPS. For the initial 2 h (pH 1.2), there was a quite controlled release followed by increased drug release. This might be due to swelling of polysaccharide in 1.2 pH followed by changing into the solubilized form in 6.8 pH and therefore revealing the pH sensitive characteristics of ALPS (Kumar & Pal, 2011). Therefore from the in-vitro drug release study, it confirms the uniqueness of ALPS by altering the release with respect to its concentration and pH sensitive characteristics. 3.8.Drug release kinetics Drug release kinetics was performed by means of Korsmeyer–Peppas, Hixson–Crowell and Higuchi models which are presented in Table 3. Drug release kinetics explains the release mechanism of drug from the solid dosage form. The tablets prepared from ALPS follows the Hixson-Crowell model and found to be the best model fit. The Hixson-Crowell model explains the release of drug from the systems where there is a change in surface area and diameter of tablets. As evident from the in-vitro drug release, the tablets prepared from ALPS tend to swell initially and followed by disintegrating into individual granular particles. So there was a decrease in the surface area of the tablets with respect to dissolution time, hence following Hixson-Crowell model.
4. Conclusion Water soluble polysaccharide was isolated from the seeds of Albizia lebbeck L. The physicochemical properties were found to be distinct and the micromeritic properties revealed a fair flow of polysaccharide. Moreover, the foaming properties are good enough as natural foam stabilizer in food processing. Molecular weight of ALPS was found to be 1.98 × 102 kDa and contains Mannose (4.06%), rhamnose (22.79%), glucose (38.9%), galactose (17.84%) and xylose (16.42%). Morphological studies showed an irregular particles with both rough and smooth surface. The FTIR analysis confirms the polysaccharide nature. Thermal studies reveals the degraradation of polymer occurred at higher temperatures (243-340°C) resulting in depolymerisation and suggests the ALPS as a thermally stable polymer. From the zetapotential measurement, the ALPS was found as non-ionic polysaccharide. The drug release profile and kinetics suggests that ALPS can be used as pH senstive polymer for the contolled release of drugs. Acknowledgement We sincerely acknowledge Central instrumentation facility, B.I.T Mesra for providing all facilities for instrumental analysis. References Abdul-Hamid, A., & Luan, Y. S. (2000). Functional properties of dietary fibre prepared from defatted rice bran. Food Chemistry, 68(1), 15–19. Ahmed, M. J., & Theydan, S. K. (2014). Optimization of microwave preparation conditions for activated carbon from Albizia lebbeck seed pods for methylene blue dye adsorption. Journal of Analytical and Applied Pyrolysis, 105, 199–208. Al-Massarani, S. M., El Gamal, A. A., Abd El Halim, M. F., Al-Said, M. S., Abdel-Kader, M. S., Basudan, O. A., & Alqasoumi, S. I. (2016). New acyclic secondary metabolites from the
biologically active fraction of Albizia lebbeck flowers. Saudi Pharmaceutical Journal, 25(1), 110–119. Bagchi, S., & Jayaram Kumar, K. (2016). Studies on water soluble polysaccharides from Pithecellobium dulce (Roxb.) Benth. seeds. Carbohydrate Polymers, 138, 88–93. Ben, M., Haddar, A., Ghazala, I., Ben, K., & Boisset, C. (2017). Optimization of polysaccharides extraction from watermelon rinds : Structure, functional and biological activities. Food Chemistry, 216, 355–364. Ben, R., Kolsi, A., Fakhfakh, J., Krichen, F., Jribi, I., Chiarore, A., … Belghith, K. (2016). Structural characterization and functional properties of antihypertensive Cymodocea nodosa sulfated polysaccharide. Carbohydrate Polymers, 151, 511–522. Bobby, M. N., Wesely, E. G., & Johnson, M. (2012). High performance thin layer chromatography profile studies on the alkaloids of Albizia lebbeck. Asian Pacific Journal of Tropical Biomedicine, 2, S1–S6. Chien, R. C., Yen, M. T., Tseng, Y. H., & Mau, J. L. (2015). Chemical characteristics and antiproliferation activities of Ganoderma tsugae polysaccharides. Carbohydrate Polymers, 128, 90–98. Deepika, V., Jayaram Kumar, K., & Anima, P. (2013). Isolation and physicochemical characterization of sustained releasing starches from Dioscorea of Jharkhand. International Journal of Biological Macromolecules, 55, 193–200. Gupta, R. S., Kachhawa, J. B. S., & Chaudhary, R. (2006). Antispermatogenic, antiandrogenic activities of Albizia lebbeck (L.) Benth bark extract in male albino rats. Phytomedicine,
13(4), 277–283. Khan, M. Z., Stedul, H. P., & Kurjaković, N. (2000). A pH-dependent colon-targeted oral drug delivery system using methacrylic acid copolymers. II. Manipulation of drug release using Eudragit L100 and Eudragit S100 combinations. Drug Development and Industrial Pharmacy, 26(5), 549–54. Kosaraju, S. L. (2005). Colon targeted delivery systems: review of polysaccharides for encapsulation and delivery. Critical Reviews in Food Science and Nutrition, 45(4), 251–8. Kulkarni, S. D., Sinha, B. N., & Jayaram Kumar, K. (2013). Synthesis, characterization and evaluation of release retardant modified starches of Lagenaria siceraria seeds. International Journal of Biological Macromolecules, 61, 396–403. Kumar Varma, C. A., Panpalia, S. G., & Kumar, K. J. (2014). Physicochemical and release kinetics of natural and retrograded starch of Indian palmyrah shoots. International Journal of Biological Macromolecules, 66, 33–39. Kumar, A., & Pal, D. (2011). Development of pH-sensitive tamarind seed polysaccharide – alginate composite beads for controlled diclofenac sodium delivery using response surface methodology. International Journal of Biological Macromolecules, 49(4), 784–793. Lam, S. K., & Ng, T. B. (2011). First report of an anti-tumor, anti-fungal, anti-yeast and antibacterial hemolysin from Albizia lebbeck seeds. Phytomedicine, 18(7), 601–608. Liu, J., Meng, C. guang, Yan, Y. hua, Shan, Y. na, Kan, J., & Jin, C. hai. (2016). Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide. International Journal of Biological Macromolecules, 82, 719–724.
Missanjo, E., Maya, C., Kapira, D., Banda, H., & Kamanga-Thole, G. (2013). Effect of Seed Size and Pretreatment Methods on Germination of Albizia lebbeck. ISRN Botany, 2013, 1–4. Mokni Ghribi, A., Sila, A., Maklouf Gafsi, I., Blecker, C., Danthine, S., Attia, H., ... Besbes, S. (2015). Structural, functional, and ACE inhibitory properties of water-soluble polysaccharides from chickpea flours. International Journal of Biological Macromolecules, 75, 276–282. Munir, H., Shahid, M., Anjum, F., & Mudgil, D. (2016). Structural, thermal and rheological characterization of modified Dalbergia sissoo gum-A medicinal gum. International Journal of Biological Macromolecules, 84, 236–245. Nandan, C. K., Sarkar, R., Bhanja, S. K., Sikdar, S. R., & Islam, S. S. (2011). Isolation and characterization of polysaccharides of a hybrid mushroom ( backcross mating between PfloVv12 and Volvariella volvacea ). Carbohydrate Research, 346(15), 2451–2456. Pang, Z., Deeth, H., & Bansal, N. (2015). Effect of polysaccharides with different ionic charge on the rheological, microstructural and textural properties of acid milk gels. Food Research International, 72, 62–73. Shahidi, F., Han, X. Q., & Synowiecki, J. (1995). Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chemistry, 53, 285–293. Shaikh, F. K., Gadge, P. P., Shinde, A. A., Padul, M. V., & Kachole, M. S. (2014). Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck) that inhibits the growth of cotton bollworm (Helicoverpa armigera). Journal of Asia-Pacific Entomology, 17(3), 319–325. Sun, H. H., Mao, W. J., Chen, Y., Guo, S. D., Li, H. Y., Qi, X. H., … Xu, J. (2009). Isolation,
chemical characteristics and antioxidant properties of the polysaccharides from marine fungus Penicillium sp. F23-2. Carbohydrate Polymers, 78(1), 117–124. Tan, H. F., & Gan, C. Y. (2016). Polysaccharide with antioxidant, α-amylase inhibitory and ACE inhibitory activities from Momordica charantia. International Journal of Biological Macromolecules, 85, 487–496. United States Pharmacopeial Convention, USP 31. (2008). The United States pharmacopeia; NF 26: The National formulary. Rockville: United States Pharmacopeial Convention. Wang, Y., Huang, M., Sun, R., & Pan, L. (2015). Extraction, characterization of a Ginseng fruits polysaccharide and its immune modulating activities in rats with Lewis lung carcinoma. Carbohydrate Polymers, 127, 215–221. York, W. S., Darvill, A. K., McNeil, M., Stevenson, T. T., & Albersheim, P. (1985). Isolation and characterization of plant cell walls and cell wall components. Methods in Enzymology, 118, 33–40.
Figure captions Fig. 1. A) Gel permeation chromatogram of ALPS using Sepharose 6B column Fig. 2. HPLC analysis of A) monosaccharide standards and B) ALPS Fig. 3. A) FTIR spectrum B) Thermogram of Albizia lebbeck L. seed polysaccharide Fig. 4. Scanning electron micrographs (A-250 X and B-750 X) of Albizia lebbeck L. seed polysaccharide. Fig. 5. In-vitro drug release profile of diclofenac sodium tablets prepared from 5, 7.5 and 10 % ALPS.
Fig. 1.
Mannose
Rhamnose
Galactose
Glucose
Arabinose
Galactose Rhamnose Mannose
Fig. 2.
Glucose
Xylose
Xylose
A Fucose
B
A
B
Fig. 3.
A
B
Fig. 4 pH 6.8
pH 1.2
%Cumulative drug release
110
90
70 ALPS 5% 50 ALPS 7.5% 30 ALPS 10% 10
-10
Fig. 5.
0
60
120
180
240
300
Time (Mins)
360
420
480
Table 1. Foaming capacity and stability of ALPS at different concentrations (1, 2, 3, 4 & 5%) Concentration (% w/v) Parameter
Time (Min) 1
2
3
4
5
FC (%)
0
30.00 ± 1.14
44.44 ± 1.24
75.00 ± 0.54
87.50 ± 1.88
87.50 ± 0.76
FS (%)
10
20.00 ± 2.52
33.33 ± 1.42
62.50 ± 1.72
87.50 ± 1.12
87.50 ± 0.58
20
15.00 ± 1.26
22.22 ± 1.80
62.50 ± 1.68
62.50 ± 1.64
87.50 ± 0.62
30
10.00 ± 0.84
11.11 ± 1.82
50.00 ± 1.28
62.50 ± 1.52
87.50 ± 0.88
60
10.00 ± 1.68
11.11 ± 0.64
37.50 ± 2.12
62.50 ± 0.94
87.50 ± 0.52
All values represent the means of triplicate analysis ± standard deviation.
Table 2. Micromeritic properties of ALPS from Albizia lebbeck L. seeds. Sample Bulk density
ALPS
Carr’s index (%)
Tapped density
Hausner
(g/ml)
(g/ml)
ratio
0.34 ± 0.06
0.42 ± 0.11
1.22 ± 0.09 17.82 ± 0.15
All values represent the means of triplicate analysis ± standard deviation.
Angle of repose (°)
Porosity
True density (g/ml)
33.73 ± 0.12
1.58 ± 0.19
1.09 ± 0.07
Table 3. Evaluation properties of Diclofenac sodium tablets prepared from ALPS with different concentrations (5, 7.5 and 10 % w/w). Sample
Average weight (mg)
Thickness (mm)
Diameter (mm)
Hardness (kg/cm2)
Friability (%)
Drug content (%)
Korsmeyer &Peppas equation n
K
r2
Hixson Crowell
Higuchi equation
k
r2
k
r2
ALPS 5%
250.10±0.09
2.39±0.04
3.99±0.02
5.64±0.34
0.69
99.68
0.79
1.065
0.9257
0.001
0.9534
4.305
0.8645
ALPS 7.5%
251.30±0.11
2.45±0.01
3.98±0.01
5.78±0.48
0.82
98.94
0.70
1.420
0.9227
0.002
0.9560
4.604
0.8757
ALPS 10%
249.60±0.16
2.24±0.04
3.99±0.01
5.89±0.16
0.74
99.98
0.61
2.659
0.8985
0.002
0.9557
4.936
0.8826
All values represent the means of triplicate analysis ± standard deviation.
S0144-8617(17)30884-6 http://dx.doi.org/doi:10.1016/j.carbpol.2017.08.017 CARP 12624
To appear in: Received date: Revised date: Accepted date:
6-7-2017 25-7-2017 3-8-2017
Please cite this article as: Kumar Varma, Chekuri Ashok., & Jayaram Kumar, K., Structural, functional and pH sensitive release characteristics of water-soluble polysaccharide from the seeds of Albizia lebbeck L.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.08.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural, functional and pH sensitive release characteristics of water-soluble polysaccharide from the seeds of Albizia lebbeck L. Chekuri Ashok Kumar Varma1, K. Jayaram Kumar1* 1
Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra,
Ranchi-835215, Jharkhand, India. *Corresponding author. Tel.: +91 06512276247; fax: +91 06512275290. E-mail addresses: [email protected] (K. Jayaram Kumar)
Highlights
Mainly comprises of glucose, rhamnose, galactose, xylose and mannose.
High swelling, water holding and foaming capacity were observed.
ALPS was found to be a non-ionic polysaccharide.
ALPS exhibited pH sensitive drug release.
Abstract Plant polysaccharides, generally regarded as safe (GRAS), are gaining importance as excipients in drug delivery. Therefore, the current paper presents the studies on structural, functional and drug release study of water soluble polysaccharide (ALPS) from seeds of Albizia lebbeck L. High swelling, water holding capacity, foam stability and lower moisture content suggests its use as additive in food preparations. The apparent molecular weight of polysaccharide was found to be 1.98 × 102 kDa. Monosaccharide composition analysis indicated that ALPS consists of mannose (4.06%), rhamnose (22.79%), glucose (38.9%), galactose (17.84%) and xylose (16.42%). Micromeritic properties revealed that the polysaccharide possess potential for pharmaceutical applications. From the surface charge analysis, ALPS was found to be non-ionic polysaccharide. Morphological study reveals the polysaccharide with irregular particle shape and rough surface. Fourier transformed infrared spectroscopy (FTIR) study confirms the carbohydrate nature of polysaccharide. From the thermogravimetric analysis (TGA) data, the second mass loss (243340oC) attributed to polysaccharide degradation. The drug release profile reveals the use of polysaccharide for the preparation of pH sensitive pharmaceutical dosage forms. Key words: water soluble polysaccharide, FTIR, pH sensitive.
1. Introduction Polysaccharides, one of the major class of biopolymers which are widely used in almost all sectors. As these polysaccharides are fermented by the colonic microflora, they falls into the category of GRAS (Kosaraju, 2005). Even though, there are large number of polysaccharides available in market but currently an extensive research is going on to explore the new sources of polysaccharides. This is due to huge demand of polysaccharides with desired characteristics in the market. These polysaccharides has a tremendous use in pharmaceutical sector such as binder, disintegrant, thickening and stabilizing agent (Kulkarni, Sinha, & Jayaram Kumar, 2013). These are also having tremendous role in targeting and controlled release of drugs. Water soluble polysaccharides, which are more preferable in the pharmaceutical sector, as they are easy to handle with regard to its high solubility and hydrophilic nature. Due to this peculiar characteristics of water soluble polysaccharide, further physical or chemical modification process makes easy to obtain the desired characteristics of polymer (Munir, Shahid, Anjum, & Mudgil, 2016). From the point of drug delivery, few drugs requires targeting to particular site either for the absorption from gastrointestinal (GI) system or for site specific diseases. The pH-dependent systems are generally accepted to fulfill this requirement, as the pH of GI tract increases from stomach (pH 2-3) to colon (7-8) (Khan, Stedul, & Kurjaković, 2000). Taking this advantage, natural pH sensitive polysaccharides without any tailored made process are need to be explored. Albizia lebbeck L., the Indian siris, flea tree or fry wood, belonging to the family Leguminosae, Mimosoideae (Al-Massarani et al., 2016; Shaikh, Gadge, Shinde, Padul, & Kachole, 2014). It is a perennial tree, which is widely distributed in India and adopted to other tropical and sub-tropical regions (Al-Massarani et al., 2016; Lam & Ng, 2011). It produces a seed pods with a length of 1530 cm and contains 6-12 seeds (Bobby, Wesely, & Johnson, 2012; Missanjo, Maya, Kapira, Banda,
& Kamanga-Thole, 2013). The seeds are consumed for the treatment of piles, diarrhea and scrofulous swelling, whereas the seed oil is used for the treatment of leprosy (Bobby, Wesely, & Johnson, 2012; Gupta, Kachhawa, & Chaudhary, 2006). These pods comprises of cellulose (36.4%), hemicellulose (8.9%), lignin (3.6%), and volatile matter (83.1%) (Ahmed & Theydan, 2014). As the seeds are rich in polysaccharides, the current study focuses on exploration of water soluble polysaccharide from Albizia lebbeck L. seeds and to characterize further in terms of structural, functional and morphological characteristics. Finally, evaluation of the isolated polysaccharide for tablet preparation and to study its drug release profile. 2. Materials and methods 2.1. Materials Albizia lebbeck L. pods were collected from B.I.T. Mesra, Ranchi and authenticated at botanical survey of India, Kolkata with specimen no. AL-01. 3- Methyl-1-phenyl-2-pyrazoline-5-one (PMP) was purchased from Sigma Aldrich. Sugar standards (Arabinose, Glucose, galactose, xylose, fucose, rhamnose and mannose) were purchased from Hi-Media. All other reagents used were of analytical grade. 2.2. Isolation and purification of Polysaccharide The pods of Albizia lebbeck L. were dehusked to obtain the seeds, which were then washed with distilled water. The seeds were then steeped in distilled water for 24 h. The imbibed seeds were then boiled for 8 h and the obtained slurry was kept at 4-8 oC for 12 h, followed by centrifugation at 5000 RPM. The resulted supernatant fluid was then precipitated in 95 % ethanol. Further, the suspension was centrifuged and then dialyzed with distilled water for 24 h. Finally, the dialyzed suspension was lyophilized and further purified by using gel permeation chromatography containing Sepharose-6B column (90cm x 2.1cm) using deionized water as eluent with a flow rate
of 0.4 ml/sec (Nandan, Sarkar, Bhanja, Sikdar, & Islam, 2011). 4 ml of eluent was collected in each test tubes and measured spectrophotometrically by using phenol-sulphuric acid method at 245 nm (York, Darvill, McNeil, Stevenson, & Albersheim, 1985). A plot of number of test tubes versus absorbance was shown in Fig. 1. The homogeneous fraction was collected with a test number ranging from 16-21 and lyophilized to yield water soluble polysaccharide (ALPS). Molecular weight determination Gel-chromatographic technique was used to determine the molecular weight of polysaccharide by using standard dextrans (T-40, T-60, T-70, T-200 & T-250) (Nandan, Sarkar, Bhanja, Sikdar, & Islam, 2011). Initially, the standard dextrans were passed through Sepharose-6B column and the elution volumes were plotted against their respective molecular weights. The apparent molecular weight of ALPS was determined by comparing the elution volume with the standard dextrans.
2.3. Monosaccharide composition analysis Monosaccharide composition was determined by means of reverse phase high pressure liquid chromatography using pre-column derivatization process (Sun et al., 2009). To 1 ml of 0.5mg/ml polysaccharide, 1 ml of 4M trifluoroacetic acid (TFA) was added and heated on a water bath at 120 oC for 2 h. Further, the excess of TFA was removed by co-distillation with methanol. To the dry hydrolysate, 1 ml of NaOH and 1ml of 0.5 M methanolic PMP was added. Then the mixture was incubated at 70 oC for 1 h and then cooled, followed by addition of 1 ml of 0.3M HCl solution. Further, the mixture was centrifuged for 5 min and the supernatant containing labelled carbohydrates were filtered through 0.22 µm nylon filter. Finally, the resulting mixture was injected into C18 - SPHERISORB column (Waters, 4.6 mm x 150 mm) connected with a PDA detector (245 nm). 0.1 M phosphate buffer (pH-6.7) -acetonitrile (83:17) was used as a mobile
phase at a flow rate of 1ml/min. The identification and quantitative determination of sugars was determined by comparison with the standard sugars (D-glucose, L-arabinose, D-xylose, L-fucose, D-galactose, L-rhamnose and D-mannose). The molar ratio of monosaccharide was calculated from the peak area. 2.4. Determination of functional properties Functional properties of isolated polysaccharide-ALPS was done by determining the moisture content, pH, swelling and solubility power, water holding capacity and fat binding capacity. The pH of 1 % ALPS solution was determined by using digital pH meter. 2.4.1. Water-holding capacity (WHC) WHC was determined according to the method described by Deepika, Jayaram Kumar and Anima (2013) with slight modifications. A suspension of polysaccharide (1 g) in distilled water was prepared and kept under magnetic stirring for 1 h. The resulted suspension was centrifuged at 3000 rpm. The settled polysaccharide was weighed (WS) after decanting the supernatant. The WHC was calculated by the following equation; 𝑊𝑆 𝑊𝐻𝐶 = ( ) × 100 𝑊 Where W is the weight of dried polysaccharide. 2.4.2. Fat binding capacity (FBC) The fat-binding capacity was determined by the method described by Abdul-Hamid and Luan (2000). The polysaccharide suspension was prepared by suspending 0.5 g of polysaccharide in 10 ml of soybean oil and agitated for 30 min at a time interval of every 5 min. The suspension was then centrifuged at 3000 rpm for a duration of 10 min. The supernatant free oil was removed and decanted for 30 min on a filter paper. The fat binding capacity was computed by the following equation:
𝐹𝑎𝑡 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 (𝑔 𝑜𝑖𝑙/𝑔) =
𝑊𝑟 − 𝑊 𝑊
Where Wr is the weight of residual polysaccharide; W is the weight of dried polysaccharide. 2.4.3. Swelling and solubility power The swelling and solubility power of polysaccharide was determined by the method described by Kumar, Varma, and Panpalia (2014). The polysaccharide suspension was subjected to heating on a water bath at 30 °C, 60 °C, 90 °C for 30 min with occasional stirring to prevent lump formation. The suspension was allowed to cool and centrifuged at 3000 rpm for 15 min. The supernatant was transferred into a pre-weighed petri-dishes and dried for 2 h at 130 °C. The residue obtained after drying the supernatant was weighed and it represents the soluble portion of polysaccharide. The swelling power and solubility were calculated as: 𝑊𝑠𝑢 ⁄𝑊 ) × 100 𝑖
% 𝑆𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 = (
𝑊𝑠𝑠 % 𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 = ( ) × 100 𝑊𝑖 × (100 − % 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦) Wsu is the weight of soluble residue in supernatant; Wi is the weight of starch on dry basis; Wss is the weight of wet starch (g). 2.4.4. Foaming properties Foaming properties of ALPS was evaluated by determining the foam capacity (FC) and foam stability (FS) according to the method followed by Shahidi, Han and Synowiecki (1995). Different concentration of ALPS (1%, 2%, 3%, 4% and 5 % w/v) were prepared and homogenized using IKA homogenizer (Germany) for 2 min. After the homogenization process, the whipped samples were transferred into a graduated measuring cylinder. The total volume was noted before (Vo) and
after (VT) whipping. Further, the volume (Vt) was noted at 10, 20, 30 and 60 min. FC and FS was determined by the following equation: 𝐹𝐶 (%) = (
𝑉𝑇 − 𝑉𝑂 ) × 100 𝑉𝑜
𝑉𝑡 − 𝑉𝑂 𝐹𝑆 (%) = ( ) × 100 𝑉𝑜 2.5.Micromeritic properties Micromeritic properties of ALPS was determined by performing bulk and tapped density, angle of repose, Hausner ratio and Carr’s index. These properties were determined by the methods described by Varma, Panpalia and Jayaram (2014). 2.6. Morphological characterization Morphology of ALPS was determined by scanning electron microscopy (JEOL, Japan, JSM6390LV). For the analysis, the powder sample was scattered on a double sided tape which is adhered to the aluminum stub and then coated with platinum to make sample conductive. The image was captured at an accelerating voltage of 10 Kv. 2.7.IR analysis IR spectra of ALPS was obtained by using Fourier transform infra-red spectroscopy (FTIR-8400 S, Shimadzu, Japan). The sample was blended with Kbr and pressed into pellets, operated in the frequency range of 4000-400 cm-1. 2.8. Thermogravimetric analysis
Thermogravimetric analysis was performed by using Thermogravimetric analyzer (DTG-60, Shimadzu, Japan. The sample was subjected to heating (10 oC/min) in the temperature range of 30-600 oC under nitrogen environment. 2.9. Determination of surface charge Surface charge of a polysaccharide was determined by means of a zeta potential measurement, followed by the method described by Pang, Deeth and Bansal (2015). Stock solutions of ALPS was prepared in MilliQ water by magnetically stirring and heating at 80 oC for 30 min. Further, the solution was allowed to stir for 2 h at room temperature. The solution was made up to 0.1 % concentration by diluting with MilliQ water. Then the stock solutions were adjusted to different pH (2.3, 4.0, 4.3, 5.3, 6.3). Finally, the zeta potential of polysaccharide solutions were measured by using Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK). 2.10. Preparation of tablets and their evaluation The ALPS polysaccharide was used to prepare the tablets by means of wet granulation and diclofenac sodium was used as model drug. Lactose was used as diluent, magnesium stearate and talc were used as lubricants. The tablets with different concentrations of polysaccharide (5, 7.5 & 10%) were prepared and evaluated for physical tests like weight variation, hardness, friability, disintegration time, drug content and cumulative drug release. Drug release study of tablets were determined according to the method described by Varma, Panpalia and Jayaram (2014). 2.11.
Drug release Kinetics
Drug release kinetic study was performed in order explore the release behavior of drug from the tablets. The kinetic study was best explained by applying Hixson–Crowell, Higuchi’s model and Korsmeyer–Peppas equations. 2.12. Statistical analysis
All the values of analysis were reported on the averages of triplicate analysis and was expressed by means ± standard deviation. 3. Results and discussion 3.1. Physicochemical characterization of polysaccharide The percentage yield of polysaccharide was found to be 12.72 % ± 1.17 and the pH was found to be neutral. The moisture content of ALPS was found to be 10.97 %, suggesting for its use in dosage form formulations. Water holding capacity was found to be 835.65 ± 0.22 %, which is higher than that of Cymodocea nodusa sulfated polysaccharide (R. Ben et al., 2016). FBC of ALPS (4.69 ± 0.15 g oil/g sample) was also found to be significantly higher than that of chickpea flour (3.15 g oil/g sample) (Mokni Ghribi et al., 2015). The swelling and solubility power determines the controlled drug releasing behavior of polysaccharide. The swelling power and solubility power of ALPS was found to be 6.09 ± 0.19 % and 83.6 ± 0.15%, which is higher than that of polysaccharide isolated form Pithecellobium dulce seeds (Bagchi & Jayaram Kumar, 2016). Foam capacity and stability helps in determining the interfacial properties of surfactants used in the formulation containing two immiscible phases. The foaming properties of ALPS were measured at different concentration from 1 to 5% and the results were shown in Table 1. At 0 min, the foam capacity was found to be concentration dependent and found to increase up to 4% and became constant after this point. At 10 min, the foam stability reduced to a concentration range of 1-3% and further reduced for 1-4% at 20 min. The of 4 % concentration remained constant after 20 min At the same time, the foam stability of 5% remains constant throughout the study. This suggests that ALPS can be used for the formulations where there is a requirement to stabilize the interfacial forces among the liquid-gas (Mokni Ghribi et al., 2015). So, the ALPS can be used in
the food industry for the preparation of milkshakes, marshmallows, ice creams (Tan& Gan, 2016); in cosmetics such as liquid soap, shaving cream. Molecular weight and monosaccharide composition The apparent molecular weight of ALPS was determined from the elution curve obtained from sepharose 6B column in comparison with standard dextrans (T-40, T-60, T-70, T-200 & T-250) (Nandan, Sarkar, Bhanja, Sikdar, & Islam, 2011). From the elution curve of ALPS (Fig. 1), the polysaccharide was found in the 16-21 test tubes and from the calibration curve of standard dextrans, the apparent molecular weight was found to be 1.98 × 102 kDa. Monosaccharide composition analysis were performed by reverse phase chromatography and shown in Fig. 2. ALPS was found to contain Mannose (4.06%), rhamnose (22.79%), glucose (38.9%), galactose (17.84%) and xylose (16.42%) with a molar ratio of 1:5.6:9.5:4.3:4. Micromeritic properties Micromeritic properties of ALPS were mentioned in Table 2. The ALPS was found to have fair flow with a low Hausner ratio and Carr’s index. The angle of repose reveals passable flow property of ALPS. This suggest the use of ALPS in pharmaceutical sector, where there is requirement of material transfer through feeding devices. 3.2.FTIR FTIR spectrum of ALPS was presented in Fig. 3A. A broad band around 3200-3400 cm-1 represents the O-H stretching and a peak at 1650 cm-1 represents the C-O stretching (Chien, Yen, Tseng, & Mau, 2015). The peaks between 1000-2000 cm-1 represents the pyranose ring of sugar molecule. A peak at 840 and 890 cm-1 may be attributed to α- and β glycosidic linkages. The above FTIR data reveals the characteristics of a typical polysaccharide.
3.3.Thermogravimetric analysis Thermogravimetric analysis of ALPS (Shown in Fig.3B.) was performed to determine the stages of decomposition of polysaccharide with respect to increase in temperature. The ALPS showed three stages of weight loss, where the first stage involves the loss of bound moisture from the polysaccharide. The second mass loss (243-340°C) involves the degradation of polymer which corresponds to the depolymerisation of the polysaccharide structure (Liu et al., 2016). Finally, the third mass loss is related to the oxidation of organic matter. 3.4.Morphology of isolated polysaccharide Morphology of ALPS was studied by using scanning electron microscope and presented in Fig. 4. ALPS was found to have irregular particles with irregular particle shape and rough surface (Munir, Shahid, Anjum, & Mudgil, 2016). This characteristic surface implies the passable flow property of polysaccharide.
3.5.Surface charge The Zeta potential of ALPS was measured at different pH (2.3, 4.0, 4.3, 5.3, 6.3). At all these pH, ALPS showed a zeta potential around zero or slightly negative values lesser than -35mV. This results confirms that ALPS is a non-ionic polysaccharide (Pang, Deeth, & Bansal, 2015).
3.6.Tablet evaluation properties Tablets prepared by using ALPS were evaluated for their physical properties and were tabulated in Table 3. In order to meet the desired tablet characteristics, a series of tablet evaluation tests has been mentioned in USP (USP, 2008). The friability is one of the physical test of tablets, where it
determines the property of tablet to withstand the physical forces that are faced during packaging and transportation. The friability of all the tablets was found to be less than 1 % and hence meets the desired criteria. The weight uniformity and drug content of all the tablets meets the specifications mentioned in USP (USP, 2008). 3.7.In-vitro drug release study Drug release profile of tablets prepared with different concentration of ALPS was mentioned in Fig. 5. From the drug release study, it was found that with increase in the concentration of ALPS there is an increased release. The reason behind this might be due to high soluble content with increase in the concentration of ALPS. For the initial 2 h (pH 1.2), there was a quite controlled release followed by increased drug release. This might be due to swelling of polysaccharide in 1.2 pH followed by changing into the solubilized form in 6.8 pH and therefore revealing the pH sensitive characteristics of ALPS (Kumar & Pal, 2011). Therefore from the in-vitro drug release study, it confirms the uniqueness of ALPS by altering the release with respect to its concentration and pH sensitive characteristics. 3.8.Drug release kinetics Drug release kinetics was performed by means of Korsmeyer–Peppas, Hixson–Crowell and Higuchi models which are presented in Table 3. Drug release kinetics explains the release mechanism of drug from the solid dosage form. The tablets prepared from ALPS follows the Hixson-Crowell model and found to be the best model fit. The Hixson-Crowell model explains the release of drug from the systems where there is a change in surface area and diameter of tablets. As evident from the in-vitro drug release, the tablets prepared from ALPS tend to swell initially and followed by disintegrating into individual granular particles. So there was a decrease in the surface area of the tablets with respect to dissolution time, hence following Hixson-Crowell model.
4. Conclusion Water soluble polysaccharide was isolated from the seeds of Albizia lebbeck L. The physicochemical properties were found to be distinct and the micromeritic properties revealed a fair flow of polysaccharide. Moreover, the foaming properties are good enough as natural foam stabilizer in food processing. Molecular weight of ALPS was found to be 1.98 × 102 kDa and contains Mannose (4.06%), rhamnose (22.79%), glucose (38.9%), galactose (17.84%) and xylose (16.42%). Morphological studies showed an irregular particles with both rough and smooth surface. The FTIR analysis confirms the polysaccharide nature. Thermal studies reveals the degraradation of polymer occurred at higher temperatures (243-340°C) resulting in depolymerisation and suggests the ALPS as a thermally stable polymer. From the zetapotential measurement, the ALPS was found as non-ionic polysaccharide. The drug release profile and kinetics suggests that ALPS can be used as pH senstive polymer for the contolled release of drugs. Acknowledgement We sincerely acknowledge Central instrumentation facility, B.I.T Mesra for providing all facilities for instrumental analysis. References Abdul-Hamid, A., & Luan, Y. S. (2000). Functional properties of dietary fibre prepared from defatted rice bran. Food Chemistry, 68(1), 15–19. Ahmed, M. J., & Theydan, S. K. (2014). Optimization of microwave preparation conditions for activated carbon from Albizia lebbeck seed pods for methylene blue dye adsorption. Journal of Analytical and Applied Pyrolysis, 105, 199–208. Al-Massarani, S. M., El Gamal, A. A., Abd El Halim, M. F., Al-Said, M. S., Abdel-Kader, M. S., Basudan, O. A., & Alqasoumi, S. I. (2016). New acyclic secondary metabolites from the
biologically active fraction of Albizia lebbeck flowers. Saudi Pharmaceutical Journal, 25(1), 110–119. Bagchi, S., & Jayaram Kumar, K. (2016). Studies on water soluble polysaccharides from Pithecellobium dulce (Roxb.) Benth. seeds. Carbohydrate Polymers, 138, 88–93. Ben, M., Haddar, A., Ghazala, I., Ben, K., & Boisset, C. (2017). Optimization of polysaccharides extraction from watermelon rinds : Structure, functional and biological activities. Food Chemistry, 216, 355–364. Ben, R., Kolsi, A., Fakhfakh, J., Krichen, F., Jribi, I., Chiarore, A., … Belghith, K. (2016). Structural characterization and functional properties of antihypertensive Cymodocea nodosa sulfated polysaccharide. Carbohydrate Polymers, 151, 511–522. Bobby, M. N., Wesely, E. G., & Johnson, M. (2012). High performance thin layer chromatography profile studies on the alkaloids of Albizia lebbeck. Asian Pacific Journal of Tropical Biomedicine, 2, S1–S6. Chien, R. C., Yen, M. T., Tseng, Y. H., & Mau, J. L. (2015). Chemical characteristics and antiproliferation activities of Ganoderma tsugae polysaccharides. Carbohydrate Polymers, 128, 90–98. Deepika, V., Jayaram Kumar, K., & Anima, P. (2013). Isolation and physicochemical characterization of sustained releasing starches from Dioscorea of Jharkhand. International Journal of Biological Macromolecules, 55, 193–200. Gupta, R. S., Kachhawa, J. B. S., & Chaudhary, R. (2006). Antispermatogenic, antiandrogenic activities of Albizia lebbeck (L.) Benth bark extract in male albino rats. Phytomedicine,
13(4), 277–283. Khan, M. Z., Stedul, H. P., & Kurjaković, N. (2000). A pH-dependent colon-targeted oral drug delivery system using methacrylic acid copolymers. II. Manipulation of drug release using Eudragit L100 and Eudragit S100 combinations. Drug Development and Industrial Pharmacy, 26(5), 549–54. Kosaraju, S. L. (2005). Colon targeted delivery systems: review of polysaccharides for encapsulation and delivery. Critical Reviews in Food Science and Nutrition, 45(4), 251–8. Kulkarni, S. D., Sinha, B. N., & Jayaram Kumar, K. (2013). Synthesis, characterization and evaluation of release retardant modified starches of Lagenaria siceraria seeds. International Journal of Biological Macromolecules, 61, 396–403. Kumar Varma, C. A., Panpalia, S. G., & Kumar, K. J. (2014). Physicochemical and release kinetics of natural and retrograded starch of Indian palmyrah shoots. International Journal of Biological Macromolecules, 66, 33–39. Kumar, A., & Pal, D. (2011). Development of pH-sensitive tamarind seed polysaccharide – alginate composite beads for controlled diclofenac sodium delivery using response surface methodology. International Journal of Biological Macromolecules, 49(4), 784–793. Lam, S. K., & Ng, T. B. (2011). First report of an anti-tumor, anti-fungal, anti-yeast and antibacterial hemolysin from Albizia lebbeck seeds. Phytomedicine, 18(7), 601–608. Liu, J., Meng, C. guang, Yan, Y. hua, Shan, Y. na, Kan, J., & Jin, C. hai. (2016). Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide. International Journal of Biological Macromolecules, 82, 719–724.
Missanjo, E., Maya, C., Kapira, D., Banda, H., & Kamanga-Thole, G. (2013). Effect of Seed Size and Pretreatment Methods on Germination of Albizia lebbeck. ISRN Botany, 2013, 1–4. Mokni Ghribi, A., Sila, A., Maklouf Gafsi, I., Blecker, C., Danthine, S., Attia, H., ... Besbes, S. (2015). Structural, functional, and ACE inhibitory properties of water-soluble polysaccharides from chickpea flours. International Journal of Biological Macromolecules, 75, 276–282. Munir, H., Shahid, M., Anjum, F., & Mudgil, D. (2016). Structural, thermal and rheological characterization of modified Dalbergia sissoo gum-A medicinal gum. International Journal of Biological Macromolecules, 84, 236–245. Nandan, C. K., Sarkar, R., Bhanja, S. K., Sikdar, S. R., & Islam, S. S. (2011). Isolation and characterization of polysaccharides of a hybrid mushroom ( backcross mating between PfloVv12 and Volvariella volvacea ). Carbohydrate Research, 346(15), 2451–2456. Pang, Z., Deeth, H., & Bansal, N. (2015). Effect of polysaccharides with different ionic charge on the rheological, microstructural and textural properties of acid milk gels. Food Research International, 72, 62–73. Shahidi, F., Han, X. Q., & Synowiecki, J. (1995). Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chemistry, 53, 285–293. Shaikh, F. K., Gadge, P. P., Shinde, A. A., Padul, M. V., & Kachole, M. S. (2014). Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck) that inhibits the growth of cotton bollworm (Helicoverpa armigera). Journal of Asia-Pacific Entomology, 17(3), 319–325. Sun, H. H., Mao, W. J., Chen, Y., Guo, S. D., Li, H. Y., Qi, X. H., … Xu, J. (2009). Isolation,
chemical characteristics and antioxidant properties of the polysaccharides from marine fungus Penicillium sp. F23-2. Carbohydrate Polymers, 78(1), 117–124. Tan, H. F., & Gan, C. Y. (2016). Polysaccharide with antioxidant, α-amylase inhibitory and ACE inhibitory activities from Momordica charantia. International Journal of Biological Macromolecules, 85, 487–496. United States Pharmacopeial Convention, USP 31. (2008). The United States pharmacopeia; NF 26: The National formulary. Rockville: United States Pharmacopeial Convention. Wang, Y., Huang, M., Sun, R., & Pan, L. (2015). Extraction, characterization of a Ginseng fruits polysaccharide and its immune modulating activities in rats with Lewis lung carcinoma. Carbohydrate Polymers, 127, 215–221. York, W. S., Darvill, A. K., McNeil, M., Stevenson, T. T., & Albersheim, P. (1985). Isolation and characterization of plant cell walls and cell wall components. Methods in Enzymology, 118, 33–40.
Figure captions Fig. 1. A) Gel permeation chromatogram of ALPS using Sepharose 6B column Fig. 2. HPLC analysis of A) monosaccharide standards and B) ALPS Fig. 3. A) FTIR spectrum B) Thermogram of Albizia lebbeck L. seed polysaccharide Fig. 4. Scanning electron micrographs (A-250 X and B-750 X) of Albizia lebbeck L. seed polysaccharide. Fig. 5. In-vitro drug release profile of diclofenac sodium tablets prepared from 5, 7.5 and 10 % ALPS.
Fig. 1.
Mannose
Rhamnose
Galactose
Glucose
Arabinose
Galactose Rhamnose Mannose
Fig. 2.
Glucose
Xylose
Xylose
A Fucose
B
A
B
Fig. 3.
A
B
Fig. 4 pH 6.8
pH 1.2
%Cumulative drug release
110
90
70 ALPS 5% 50 ALPS 7.5% 30 ALPS 10% 10
-10
Fig. 5.
0
60
120
180
240
300
Time (Mins)
360
420
480
Table 1. Foaming capacity and stability of ALPS at different concentrations (1, 2, 3, 4 & 5%) Concentration (% w/v) Parameter
Time (Min) 1
2
3
4
5
FC (%)
0
30.00 ± 1.14
44.44 ± 1.24
75.00 ± 0.54
87.50 ± 1.88
87.50 ± 0.76
FS (%)
10
20.00 ± 2.52
33.33 ± 1.42
62.50 ± 1.72
87.50 ± 1.12
87.50 ± 0.58
20
15.00 ± 1.26
22.22 ± 1.80
62.50 ± 1.68
62.50 ± 1.64
87.50 ± 0.62
30
10.00 ± 0.84
11.11 ± 1.82
50.00 ± 1.28
62.50 ± 1.52
87.50 ± 0.88
60
10.00 ± 1.68
11.11 ± 0.64
37.50 ± 2.12
62.50 ± 0.94
87.50 ± 0.52
All values represent the means of triplicate analysis ± standard deviation.
Table 2. Micromeritic properties of ALPS from Albizia lebbeck L. seeds. Sample Bulk density
ALPS
Carr’s index (%)
Tapped density
Hausner
(g/ml)
(g/ml)
ratio
0.34 ± 0.06
0.42 ± 0.11
1.22 ± 0.09 17.82 ± 0.15
All values represent the means of triplicate analysis ± standard deviation.
Angle of repose (°)
Porosity
True density (g/ml)
33.73 ± 0.12
1.58 ± 0.19
1.09 ± 0.07
Table 3. Evaluation properties of Diclofenac sodium tablets prepared from ALPS with different concentrations (5, 7.5 and 10 % w/w). Sample
Average weight (mg)
Thickness (mm)
Diameter (mm)
Hardness (kg/cm2)
Friability (%)
Drug content (%)
Korsmeyer &Peppas equation n
K
r2
Hixson Crowell
Higuchi equation
k
r2
k
r2
ALPS 5%
250.10±0.09
2.39±0.04
3.99±0.02
5.64±0.34
0.69
99.68
0.79
1.065
0.9257
0.001
0.9534
4.305
0.8645
ALPS 7.5%
251.30±0.11
2.45±0.01
3.98±0.01
5.78±0.48
0.82
98.94
0.70
1.420
0.9227
0.002
0.9560
4.604
0.8757
ALPS 10%
249.60±0.16
2.24±0.04
3.99±0.01
5.89±0.16
0.74
99.98
0.61
2.659
0.8985
0.002
0.9557
4.936
0.8826
All values represent the means of triplicate analysis ± standard deviation.