Extraction and derivatization of Leucaena leucocephala (Lam.) galactomannan: Optimization and characterization

International Journal of Biological Macromolecules 92 (2016) 831–841

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Extraction and derivatization of Leucaena leucocephala (Lam.) galactomannan: Optimization and characterization Neeraj Mittal, Pooja Mattu, Gurpreet Kaur ∗ Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, 147002, Punjab, India

a r t i c l e

i n f o

Article history: Received 19 May 2016 Received in revised form 12 July 2016 Accepted 13 July 2016 Available online 1 August 2016 Keywords: Carboxymethylation Galactomannan Intrinsic viscosity

a b s t r a c t Water soluble gums also known as hydrocolloids are increasingly finding applications in the pharmaceutical and food industry due to their versatile functional properties. They possess considerable use in food and pharmaceutical industries as emulsifying, thickening and gelling agents. In the present investigation a heteropolysaccharide galactomannan was extracted from Leucaena leucocephala (Lam.) seeds by an aqueous method, characterized for its compositional analysis (mannose: galactose ratio), physicochemical and functional properties (solubility), and mechanical properties. The extracted gum was derivatized to form its carboxymethyl derivative and the method of its derivatization was optimized by varying the reaction parameters. The native and derivatized gum was characterized by FTIR, XRD, DSC, NMR, SEM and elemental analysis, etc. The yield of Leucaena leucocephala galactomannan (LLG) was found to be 20% (w/w). The optimized parameters for carboxymethylation reaction (degree of substitution 0.805) were found to be 6.0 g NaOH, 10.0 g MCA, at 60 ◦ C for 4 h. The physicochemical and functional characteristics of native and derivatized gum suggest its potential role in food and pharmaceutical industries. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Seed galactomannans, also referred to as seed gums, are heterogeneous, neutral polysaccharides, comprising of galactopyranosyl (Gal) and mannopyranosyl (Man) residues [1,2]. Galactomannans constitute the second largest group of storage polysaccharides in terms of their distribution in the plant kingdom and are associated with the endosperm cell wall of seeds belonging to several botanical families [3]. These find wide applications in paper, textile, pharmaceutical, cosmetics, food and oil recovery industries, owing to their good binding, thickening, gelling, emulsifying and suspending properties. The galactomannan gums from different sources usually vary in the mannose/galactose (M/G) ratio, distribution of galactose residues along the mannan backbone and in molecular weight [4]. The molar ratio of galactose to mannose varies with plant origin but is typically in the range of 1:1, 1:2, 1:3, and 1:4 for fenugreek, guar, tara, and locust bean gum, respectively. Leucaena leucocephala (Lam.) is a perennial thornless, tropical plant, with a height of around 8 m that is native to tropical America and belongs to family Fabaceae and subfamily Mimosoideae [5]. In India, it is grown in Andhra Pradesh and Gujarat states,

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (G. Kaur). http://dx.doi.org/10.1016/j.ijbiomac.2016.07.046 0141-8130/© 2016 Elsevier B.V. All rights reserved.

and is commonly known as Subabul/Kubabul. The plant has been widely used as forage, timber, firewood, fuel, gum, organic fertilizer, and raw material for pulp and paper industry, in food and pharmaceutical industry [6]. Almost all parts of Leucaena (seeds, leaves, pods and barks) have been used in food e.g. in salads and for the preparation of many dishes [7]. In addition, it also displays anti-cancer, anti-viral, anti-coagulant, anti-thrombotic, anti-inflammatory, anti-diabetic and immunostimulant properties [8,9]. The seeds are used as a substitute for coffee and the seed gum has been used as a laxative and for controlling stomach diseases. The seeds majorly comprise of galactomannan gum, although oils (unsaturated linoleic and oleic fatty acids), tannins, oxalic acid and non protein substance mimosine are also present in minor amounts [7]. The galactomannan gum extracted from Leucaena leucocephala seed endosperm is composed of linear chains of ␤(1-4)-d-mannose units substituted by single ␣-d-galactose units at O-6. The ratio of mannose to galactose of Leucaena gum is 1.3:1 [10]. However, the mannose/galactose ratio is variable among species, portions or even fractions of Leucaena. Although galactomannans possess a wide range of applications, they display certain drawbacks such as uncontrolled hydration, pH dependent solubility and they are more prone to microbial attack. Chemical derivatization can be used as an effective means for overcoming these drawbacks and can also help in improving functional properties of galactomannans [11]. Various

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derivatives (carboxymethyl, amine, sulphate) of galactomannans have been reported to improve their functional characteristics. Among all the reactions, carboxymethylation is widely employed because of ease of processing, and versatility of the product obtained. Carboxymethyl product displays better solubility and gelling properties. Carboxymethylation of galactomannans is based upon the Williamson ether synthesis in which the galactomannan is activated with aqueous sodium hydroxide [12]. Sodium hydroxide increases the nucleophilicity of free hydroxyl groups (particulary–CH2 OH) by forming alkoxides. The gum alkoxides react with monochloro acetic acid, thus resulting in carboxymethylated gums. In the present investigation, Leucaena galactomannan was extracted, characterized and an attempt was made to chemically modify Leucaena galactomannan by carboxymethylation. The four reaction parameters that play an important role in carboxymethylation process, i.e. concentration of sodium hydroxide, concentration of monochloroacetic acid, reaction temperature and reaction time were optimized. The carboxymethylation of galactomannan was carried out employing green technology and impact of reaction parameters on DS was studied. Further, the physicochemical properties of native gum and derivatized gum were also examined. 2. Materials and methods 2.1. Materials The seeds of Leucaena Leucocephala were procured from Greenfield Agro Forestry Products, Madhya Pradesh. Isopropyl alcohol was purchased from Merck India Limited, Mumbai. Galactose and mannose were purchased from Tokyo Chemicals Co. Ltd, Japan. Monochloroacetic acid, and sodium hydroxide were purchased from S.D. fine chemicals, India. All the chemicals and reagents used in the study were of analytical grade. 2.2. Extraction and purification of leucaena leucocephala galactomannan (LLG) LLG was extracted by modification of the method reported by Malviya et al. [13]. Leucaena leucocephala seeds were first washed with water to remove any surface dust, and then dried. This was followed by coarse grinding of the seeds in a mixer. The ground seeds were allowed to swell in double volume of distilled water for 24 h followed by homogenization (Remi equipments, Mumbai). The extract was filtered with the help of muslin cloth followed by centrifugation (4000 rpm). The galactomannan gum was precipitated by adding excess isopropyl alcohol (100 ml × 3) [13]. The precipitates were separated by filtration, purified by dialysis and dried in a lyophilizer (Allied Frost, Delhi). 2.2.1. Compositional analysis Protein content of gum was determined by the Folin-Lowry method. Total ash content and acid insoluble ash was determined by reported methods [14]. 2.2.1.1. Thin layer chromatography (TLC). LLG (200 mg) was boiled in 20 ml of 10% (v/v) sulfuric acid for 5 h. The solution was cooled and an excess of barium carbonate was added with continuous stirring till the pH of the solution was 7. The solution was filtered and the filtrate was evaporated on a rotary evaporator (Perfit, India) at 30–50 ◦ C under vacuum to get gum hydrolyzate (syrupy residue). The obtained residue was dissolved in 10 ml of 40% (v/v) methanol for TLC profiling. TLC study was performed on pre-coated silica gel GF 254 plates (stationary phase) using a mixture of n-butanol, isopropyl alcohol and water in the ratio of 11:6:3 as the mobile phase.

Pure galactose and mannose solutions were used as standards. After the TLC run, the plates were sprayed with 10% (v/v) sulfuric acid solution in methanol and kept in an oven at 110 ◦ C for 1 h. The Rf values for the separated spots were measured and compared with Rf values of pure galactose and mannose [15]. 2.2.1.2. Determination of mannose/galactose ratio (M/G ratio). The M/G ratio of LLG was calculated by method reported by Pawar and Lalitha [15]. Briefly, 1 ml of the galactomannan, pure galactose or mannose (0.01% w/v) was transferred into a series of test tubes 1, 2, and 3, respectively. 5 ml of anthrone reagent (in sulphuric acid) was added to each test tube and stirred properly.The absorbance of each solution was taken at 360 nm on a UV spectrophotometer (DU640 B Series Beckman, USA). The galactose and mannose content was calculated using the following formula and M/G ratio was determined. Galactose/Mannose content(%) =

25 × B S×A

(1)

where B is the reading of sample, A is the reading of pure galactose or mannose and S is the weight of gum sample. 2.2.2. Physicochemical characterization of LLG 2.2.2.1. Intrinsic viscosity and determination of molecular weight. LLG powder (1.0 g) was dispersed in 100 ml of distilled water. The intrinsic viscosity of galactomannan was determined using a Brookfield viscometer maintained at 25 ◦ C. The density of the solutions was measured using pycnometer. The relative viscosity was calculated using the following equation: ␩r =

␩ ␩s

(2)

where ␩r is the relative viscosity, ␩ is the viscosity of the gum solution (mPas), and ␩s is the viscosity of the solvent (mPas). The intrinsic viscosity was calculated from relative viscosity using the following equation: ␩r = 1 + [␩] C

(3)

where [␩] is the intrinsic viscosity and C is the concentration of LLG [16]. The molecular weight (Mv) of LLG was determined by using Mark–Houwink–Sakurada equation [17] employing k value as 80.2 × 10−6 [18]. ␩ = 80.2 × 10−6 Mv␣

(4)

where ␣ is a constant, the value of which depends upon the solvent characteristics. Mark–Houwink–Sakurada constant (␣) for LLG is reported to be 0.79 [18]. 2.2.2.2. Determination of pH and optical rotation. The pH and optical rotation of 1.0% (w/v) solution of LLG was measured using ESICO pH meter and Perkin Elmer polarimeter, respectively. The optical rotation measurements were performed in a 10 mm path length cell kept at 25 ◦ C [19]. 2.2.3. Functional properties 2.2.3.1. Solubility determination. The solubility of dried LLG powder was determined in different buffers (pH 1.2, 5.8, 6.8, and 7.4) and in distilled water. A suspension of 1.0% (w/v) LLG (20 ml) was prepared and stirred for 24 h in distilled water and buffers. The suspensions were centrifuged at 6000g for 30 min to remove any insoluble fractions. The insoluble sedimented part was carefully taken and dried in oven at 105 ◦ C till a constant weight was obtained [20,21]. The percentage solubility was determined by following a formula: %Solubility =

C 2 − C1 × 100 C2

(5)

N. Mittal et al. / International Journal of Biological Macromolecules 92 (2016) 831–841

where C1 is the weight of sedimented fraction (mg) while C2 is the weight of initial gum sample (mg). 2.2.3.2. Effective pore radius (Reff.p ). A micropipette tip (transparent, 2 ml) was filled with LLG powder and then accurately weighed (Wa ). Hexane [surface tension (␥) 18.4 mN/m] was poured dropwise on the top of the tip till the solvent drops out at the bottom of the tip [22]. The tip was reweighed (Wb ) and Reff.p was calculated using the following formula: Reff.p =

W b − Wa 2␥

2.2.3.3. Water sorption time (WST). Dried powdered LLG (250 mg) was weighed and filled into a micropipette tip (transparent, 2 ml). The tip outlet was first blocked with nylon to avoid leakage of the powder. The tip was tapped on hard surface to obtain the uniform packing. The tip was dipped into a 2–3 mm layer of phosphate buffer pH 6.8 and the time taken by the liquid to reach to the top of the powder surface was estimated as WST [23]. 2.2.3.4. Contact angle. The contact angle measurements were performed on LLG (1.0–5.0% w/v) solutions. A volume of 200 ␮l of each solution was dropped from a fixed height on a glass slide. Images were captured employing digital camera (Nikon Coolpix L29) and analyzed using “Image J” software [24]. All measurements were performed in triplicate. 2.3. Derivatization of LLG 2.3.1. Optimization of carboxymethylation method for LLG Carboxymethyl Leucaena leucocephala galactomannan (CMLLG) was synthesized by Williamson ether synthesis. LLG (2% w/v) was dispersed in distilled water and continuously stirred on a magnetic stirrer. To this dispersion, varying concentrations of sodium hydroxide (1.5–9.0 g) were added, followed by the slow addition of monochloro acetic acid. The flasks were immersed in a thermostatic water bath at a specific temperature (50–80 ◦ C) and the reaction was allowed to proceed for a desired period of time (2–5 h). Table 1 depicts the different conditions employed for derivatization. The contents of the flasks were shaken occasionally during the reaction. The excess alkali present in the dispersion was neutralized

833

with glacial acetic acid and the reaction product was precipitated by isopropyl alcohol. The precipitates (carboxymethylated derivative of gum) were dried in a lyophilizer (Allied Frost, Delhi) [11]. 2.3.2. Determination of degree of substitution (DS) The DS was determined by the method described by Eyler et al. [25]. 1.5 g of CMLLG (sodium salt) was weighed accurately and dissolved in mixture of 50 ml of 2 M HCl in methanol, by continuous stirring. The free acid (CMLLG) was precipitated with isopropyl alcohol, washed three times with 95% (v/v) ethanol and precipitates were dried in oven at 60 ◦ C. Accurately weighed 1.0 g of the free acid form of CMLLG was dissolved in 50 ml of 0.1 M NaOH solution on a magnetic stirrer. The dissolved solution was then titrated against standardized 0.1 M HCl solution using phenolphthalein as the indicator. The DS was determined using the following formula: DS = A=

0.162A 1 − 0.058A

(7)

BC − DE F

(8)

where A = Acid consumed per gram of sample, B = NaOH solution added (ml), C = Normality of NaOH, D = HCL required for titration of excess NaOH (ml), E = Normality of HCL, F = Amount of CMLLG taken, 162 = Gram molecular mass of anhydroglucose unit, 58 = Net increase in molecular mass of anhydroglucose unit for each carboxymethyl group substituted. 2.3.3. Characterization of LLG and CMLLG derivative 2.3.3.1. Fourier transform infrared (FTIR) spectroscopy. The spectral properties of powdered LLG and CMLLG were analyzed using a FTIR spectrophotometer (NICOLET iS50, Thermo scientific). The powders were mixed with KBr and a pellet was compressed. The samples were scanned over a frequency range of 4000 and 400 cm−1 [26]. 2.3.3.2. X-ray diffraction (XRD). The crystallographic analysis of LLG and CMLLG were performed by XRD. X-ray diffractogram was recorded on XPert PRO diffractometer system equipped with Cu K␣ radiations (␭ = 1.54060 Å) generated at 45 kV and 40 mA. XRD diffractogram was collected with 2 ranging from 10 to 80◦ , step size of 0.0170◦ , scan step time of 20.0271 s at room temperature (25 ◦ C). The dried, powdered samples were placed on the sam-

Table 1 Effect of reaction conditions on degree of substitution (DS) of carboxymethylated Leucaena leucocephala galactomannan (CMLLG). Batch code

NaOH (g)

MCA (g)

Molar ratio LLG:MCA:NaOH

Temperature (◦ C)

Time (h)

CMLLG1 CMLLG2 CMLLG3 CMLLG4 CMLLG5 CMLLG6 CMLLG7 CMLLG8 CMLLG9 CMLLG10 CMLLG11 CMLLG12 CMLLG13 CMLLG14 CMLLG15 CMLLG16 CMLLG17 CMLLG18 CMLLG19 CMLLG20 CMLLG21 CMLLG22

1.5 1.5 1.5 1.5 3.0 3.0 3.0 3.0 6.0 6.0 6.0 6.0 9.0 9.0 9.0 9.0 6.0 6.0 6.0 6.0 6.0 6.0

5.0 7.5 10.0 12.5 5.0 7.5 10.0 12.5 5.0 7.5 10.0 12.5 5.0 7.5 10.0 12.5 10.0 10.0 10.0 10.0 10.0 10.0

01:00.7 1:1.5:0.7 02:00.7 1:2.5:0.7 01:01.5 1:1.5:1.5 02:01.5 1:2.5:1.5 01:01:03 01:05.5 01:02:03 02:05.5 01:01:04 01:05.5 01:02:04 02:05.5 01:02:03 01:02:03 01:02:03 01:02:03 01:02:03 01:02:03

60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 50 70 80 60 60 60

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2 3 5

*

All values are expressed as mean ± SD (n = 3).

DS* 0.024 0.065 0.102 0.085 0.177 0.355 0.534 0.299 0.521 0.678 0.805 0.629 0.412 0.518 0.717 0.473 0.729 0.695 0.591 0.209 0.518 0.724

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.003 0.021 0.009 0.019 0.015 0.011 0.024 0.025 0.023 0.017 0.034 0.029 0.019 0.016 0.028 0.031 0.026 0.045 0.029 0.007 0.015 0.044

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ple stage (PW 3071/xx Bracket) and were evaluated for diffraction patterns [27]. 2.3.3.3. Differential scanning calorimetry (DSC) analysis. The thermal attributes of gum and its carboxymethylated derivative were determined by differential scanning calorimeter (EVO131, SETARAM Instrumentational France). LLG and CMLLG derivative (2.0 mg) were heated from 40 to 400 ◦ C at 10 ◦ C/min [28]. 2.3.3.4. Nuclear magnetic resonance (NMR). Nuclear magnetic resonance spectroscopy (1 H) of LLG and CMLLG was observed using NMR spectrometer (Bruker Avance III, 400 MHz) at 25 ◦ C using D2 O as a solvent [29]. 2.3.3.5. Elemental analysis. Elemental analysis was performed for estimation of carbon, hydrogen and oxygen content in powdered native gum and its carboxymethyl derivative using CHNSO FLASH 2000 (Thermo scientific) [30]. 2.3.3.6. Zeta potential. 1.0% (w/v) solutions of LLG and CMLLG were prepared and zeta potential was determined at 25 ◦ C using Beckman coulter DelsaTM Nano. The solutions of native gum and its carboxymethylated derivative were prepared in triple distilled water and the dispersion of each sample was taken in zeta cell and zeta potential was measured [31]. 2.3.3.7. Scanning electron microscopy (SEM). The surface characteristics of the LLG and CMLLG were studied by scanning electron microscopy (JEOL, JSM- 6510LV). Powders were taken and coated with gold (auto fine coater JFC-1600) to make them conductive. Images were taken at acceleration voltages of 5–10 kV electron beam [21]. 2.3.3.8. Rheological measurements. The viscosity of LLG and CMLLG solutions (1.0–7.0% w/v) was determined using Brookfield viscometer (Model LVDV-I) using spindle no. S18. The temperature was maintained at 25 ◦ C by circulating water bath. The solutions of different concentrations (1.0–7.0% w/v) were prepared by dissolving the required amount of gum in distilled water under continuous stirring for 24 h. The solutions were centrifuged for 25 min at 25 ◦ C to remove any insoluble fractions. Evaluations were conducted in triplicate [32]. 2.3.3.9. Swelling studies. The swelling of LLG and CMLLG was determined in distilled water and in various buffers (pH 1.2, 6.8 and 8.0). Gums (100 mg) were dispersed in 50 ml of distilled water or buffer systems. The dispersions were kept for 2 h to swell properly. The supernatant was discarded and gum was recovered. Excess water or buffer was removed carefully and gums were reweighed [11,21]. The swelling index was calculated using following formula:

1.0 mm s−1 , test speed of 2.0 mm s−1 at a distance of 50 mm above the top of the sample to a depth of 10 mm, and returned back to the starting position. Firmness [g], consistency [g.s], and cohesiveness [g] was determined for six replicates of samples (30 ml). Cone penetration was determined employing spreadability rig (HDP/SR, STABLE Micro Systems). It consisted of a 45◦ conical Perspex probe (P/45C) that penetrated a conical sample holder containing sample. The probe was allowed to penetrate into the product to a distance of 50 mm at a rate of 3 mm s−1 and the work required to accomplish penetration was calculated from the area under the curve (work of shear [g.s]) [33]. 3. Results and discussion 3.1. Extraction of LLG The aqueous extraction technique used for the isolation of galactomannan from the seeds of Leucaena leucocephala resulted in the yield of 20% (w/w) of galactomannan. The reported yield of galactomannan is 25% (w/w) [34,35]. 3.1.1. Compositional analysis The protein content of LLG was found to be 2.1 ± 0.2% (w/w). The percentage total ash and acid insoluble ash value of LLG was found to be 5.01 ± 0.07% (w/w) and 0.60 ± 0.04% (w/w), respectively. The ash values reflect the adulteration levels of natural polymers. Total ash is normally composed of inorganic mixtures of carbonates, phosphates, silicates and silica. The total ash value for LLG was low, indicating low levels of contamination [36]. The TLC profile of gum hydrolyzate showed two spots on the plate. The spots were evaluated as galactose and mannose by comparing their Rf values with pure galactose and mannose. Rf value was found to be very close to that observed with galactose and mannose. The ratio of galactose and mannose varies in galactomannans. This ratio affects the solubility and rheological properties of galactomannans. The M/G ratio of LLG was found to be 1.19:1 against a reported value of 1.3:1 [10]. 3.1.2. Physicochemical characterization of LLG 3.1.2.1. Intrinsic viscosity and molecular weight of LLG. The intrinsic viscosity and viscosity average molecular weight (MV ) of LLG were found to be 3.07 ± 0.04 dL/g and 6.3 × 105 g/mol, respectively. The reported values of intrinsic viscosity and MV for LLG are 3.5 dL/g and 6.98 × 105 g/mol, respectively, which are almost similar to the observed values [18].

where W2 was the final weight and W1 was the initial weight of the galactomannan.

3.1.2.2. pH and optical rotation of LLG. The pH of the LLG solution (1.0% w/v) was found to be 6.78 ± 0.02. The near neutral pH implies that it will not cause irritation, when used in formulations and can be accepted as neutral excipient in various pharmaceutical formulations. The optical rotation of LLG was found to be +110 at 25 ◦ C. The reported value for guar gum is +68.16 i.e. dextrorotatory nature of galactomannans [26].

2.3.4. Mechanical properties The mechanical properties of LLG and CMLLG were determined employing Texture analyzer (TA-XT plus Texture Analyzer). The firmness and cohesiveness of LLG and CMLLG solution were determined by back extrusion. A rig consisting of flat 35 mm diameter disc probe (model A/BE, stable Micro systems) was driven into a large cylinder sample holder (50 mm diameter) to force down into the sample. The movement of the probe forced the sample to flow upward through the concentric annular space between the probe and the container. The probe was allowed to fall at pretest speed of

3.1.3. Functional properties 3.1.3.1. Solubility of LLG. LLG was found to be freely soluble in distilled water and different buffer systems. The solubility data of LLG revealed that there was no significant difference in solubility profile of LLG in different buffer systems (Table 2). LLG was found to be completely soluble in cold water. The solubility of galactomannans depends on the mannose-galactose ratio, higher the galactose substitution in galactomannans, the more is the solubility. The higher substitution facilitates inter-hydrogen bonding rather than intrahydrogen bonding between mannose units resulting in solubility

Swellingindex =

W2 W1

(9)

N. Mittal et al. / International Journal of Biological Macromolecules 92 (2016) 831–841 Table 2 Physicochemical properties of LLG. Parameters

Observed values for LLG

Effective pore radius (mm) Water sorption time (s) pH

0.024 ± 0.06 215 6.78 ± 0.02

Solubility (%) pH 1.2 pH 5.8 pH 6.8 pH 7.4 pH 10.0 Distilled water Intrinsic viscosity (dL/g) Molecular weight (g/mol) Mannose/Galactose ratio Protein content (%) Total ash (% w/w) Acid insoluble ash (% w/w)

78.78 ± 1.70 79.92 ± 1.84 81.41 ± 1.60 83.92 ± 1.28 80.72 ± 1.51 90.15 ± 2.34 3.07 ± 0.04 6.3 × 105 1.19:1 2.1 ± 0.2 5.01 ± 0.07 0.60 ± 0.04

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sodium hydroxide (NaOH) leading to the formation of alkoxide. The alkoxide further reacts with monochloro acetic acid (MCA) resulting in CMLLG. Fig. 1 depicts the reaction involved in derivatization of the LLG. 3.2.2. Optimization of reaction conditions for derivatization 3.2.2.1. Effect of NaOH. The DS of LLG was found to be a function of NaOH concentration at low levels. With an increase in the amount of NaOH from 1.5 g to 6.0 g the value of DS increased from 0.024 ± 0.003 to 0.521 ± 0.023 when MCA concentration was kept constant (5.0 g) as depicted in Fig. 2a and Table 1. The same pattern was observed when 7.5, 10.0 and 12.5 g MCA was used as etherifying agent. However, a further increase in the amount of NaOH to 9.0 g led to a decrease in DS. In a carboxymethylation process, two competitive reactions occur simultaneously. The first step (main step) involves reaction of hydroxyl groups of galactomannan with NaOH and MCA to form CMLLG. However, there is a side reaction that results in the formation of sodium glycolate as depicted in the following equation [30,40]

All values are expressed as mean ± SD (n = 3).

even in cold water [37]. The ratio of M/G in LLG was found to be 1.19:1 which facilitated higher solubility of the LLG. 3.1.3.2. Reff.p and WST. Reff.p of the powder depicts the porosity of powder. A higher value of Reff.p represents higher porosity of powder. Reff.p of LLG was found to be 0.024 ± 0.06 mm. WST measures the disintegration potential of powdered gum. The more is the time taken by gum to absorb the medium through the pores present in between gum particles, the lower is the disintegration potential of gum. The WST of LLG was found to be 215 s which indicated poor wicking properties of the LLG. These properties support the suitability of the gum during storage and its use in the presence of moisture sensitive ingredients [33]. 3.1.3.3. Contact angle. Contact angle measurements were performed to determine the surface wettability and hydrophobicity of the LLG. A value of contact angle less than 90◦ indicates that wetting of the surface is favorable, and the fluid will spread over a large surface area; while contact angles greater than 90◦ generally means that wetting of the surface is unfavorable so the fluid will minimize its contact with the surface and form a compact liquid droplet [38]. The contact angle of LLG solutions (1–5%w/v) was found to lie in the range of 20.15–35.21◦ . The contact angle was found to depend on polymer concentration. An increase in polymer content leads to an increase in viscosity of formulation, thus leading to increase in contact angle [39]. 3.2. Optimization and characterization of CMLLG 3.2.1. Carboxymethylation of LLG Carboxymethylation of LLG was done by aqueous method employing Williamson synthesis. The reaction proceeded in two steps: the first step involves the activation of primary alcohol with

NaOH + ClCH2 COONa

HOCH2 COONa + NaCl

This side reaction is slower than the main reaction, but in the presence of excess NaOH, the glycolate formation predominates over the main reaction and hence DS value decreases [40]. 3.2.2.2. Effect of MCA. The effect of MCA on DS is depicted in Fig. 2b.The DS of CMLLG increased when the amount of MCA was increased from 5.0 to 10.0 g (Table 1). A further increase in amount of MCA decreased the DS value. NaOH provides an alkaline environment for the carboxymethylation reaction, and also acts as swelling agent to facilitate the diffusion and penetration of MCA. The presence of excess amount of MCA reduces the amount of NaOH that can react with LLG. In addition, the glycolate formation increases with an increase in MCA amount. Similar results have been reported in carboxymethylation of tamarind kernel powder gum [41]. 3.2.2.3. Effect of temperature. The effect of temperature on the carboxymethylation of LLG is depicted in Fig. 2c. It was observed that the DS increased from 0.729 ± 0.026 to 0.805 ± 0.034 as the reaction temperature increased from 50 to 60 ◦ C and further decreased with increase intemperature (80 ◦ C) (Table 1). The increase in temperature increased the solubility of MCA and also facilitated the swelling of gum and diffusion of reactants [42]. The reaction rate increases since the proportion of molecules that have higher energy than activation energy increases [43]. A decreased in DS value at 80 ◦ C could be attributed to the higher glycolate formation. 3.2.2.4. Effect of time. Time is an important parameter in the reaction process. Fig. 2d shows the effect of reaction time on DS when 6.0 g NaOH and 10.0 g MCA were used at temperature 60 ◦ C. The DS increases with an increase in time, when the time was increased from 2 to 4 h. The DS value of CMLLG increased from 0.209 ± 0.007 to 0.805 ± 0.034 (Table 1). A decrease of DS was observed on

Fig. 1. Derivatization of LLG.

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Fig. 2. Effect of a) NaOH, b) MCA, c) temperature and d) time on degree of substitution.

increasing the time after that. The increase in DS is a direct aftermath effect of time on swelling of LLG because of the diffusion and adsorption of reactants followed by better contact between etherifying agent and LLG [41,43]. From the above observations, it was concluded that the maximum value of DS was obtained when 6.0 g NaOH and 10.0 g of MCA were used. The optimized temperature and time of reaction was found to be 60 ◦ C and 4 h, respectively.

bonds maintain the stability of gum crystals [40]. In galactomannans the intermolecular hydrogen bonding is responsible for the crystallinity of gums; the crystallinity reduces when this interaction is disrupted [45]. The crystalline size for LLG was calculated by Debye Scherrer equation which was found to be 73.11 nm.

3.2.3. Characterization of LLG and CMLLG derivative 3.2.3.1. FTIR spectroscopy. The FTIR spectra of LLG showed sharp peaks at 3274 cm−1 that could be attributed to stretching of hydroxyl groups of aliphatic alcohol as depicted in Fig. 3a. The CH-stretching vibration of alkane was observed at 2923 cm−1 , the bending vibrations of ether (C O C) groups were observed at 1018 cm−1 . All these peaks were in agreement with peaks reported by Huang et al. [44] for FTIR spectra of guar gum. The FTIR spectra of CMLLG also demonstrated similar peaks (Fig. 3b). However, intense peak was not observed at 3277 cm−1 indicating that hydroxyl groups of aliphatic alcohol were utilized in carboxymethylation. Further an additional peak at 1408 cm−1 was observed in FTIR spectra of CMLLG. This peak can be attributed to the carboxylate ion of carboxymethyl group substituted on LLG. Similar peaks have been reported in the FTIR spectra of carboxymethyl guar gum [11,44].

3.2.3.3. DSC studies. The thermal characteristics of LLG (Fig. 3c) and CMLLG (Fig. 3d) were studied by DSC. The DSC spectra of LLG showed a broad endothermic peak at 80 ◦ C which can be ascribed to the loss of water present in LLG. The melting peak of LLG was observed at 310 ◦ C. The melting point of LLG determined using melting point apparatus (Perfit melting point apparatus) was found to be 308 ◦ C. The DSC spectra of CMLLG showed an endothermic transition at 80 ◦ C and exothermic peak at 290 ◦ C. The shifting of exothermic could be attributed to the low stability of galactomannan due to carboxymethylation. The intermolecular and intramolecular hydrogen bonds are broken because of carboxymethylation. A decrease in the value of melting temperature with a decrease in H value indicates a decrease in thermal stability of gum. The shifting of exothermic peak with change in enthalpy (H) from 80.89 Jg−1 to 30.31 Jg−1 could be attributed to derivatization of LLG. Similar shift in peak temperatures and H values of carboxymethyl derivative of fenugreek gum have also been reported by Bassi and Kaur [46].

3.2.3.2. X-ray diffraction (XRD). XRD is widely employed to determine the percentage crystallinity, crystallite size, and orientation of crystallites. The diffractogram of LLG (Fig. 4a) reveals some degree of crystallinity, however,the degree of crystallinity was reduced in CMLLG (Fig. 4b). This could be attributed to the replacement of hydroxyl groups by carboxymethyl groups because hydrogen

3.2.3.4. NMR. 1 H NMR analysis of purified gum revealed peaks at ı 4.935 and 4.701 ppm due to the presence of H-1 (␣) (␣ −D- sugar units) and H-2 (␤) (␤-d- sugar units), respectively. Further, the peaks at ı 3.977, 3.869, 3.750 and 3.674 ppm corresponded to H2 to H6 . Similar peaks have been reported in literature for galactomannans [15]. The NMR spectra of CMLLG revealed the occurrence of

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Fig. 3. FTIR spectra of a) LLG, b) CMLLG and DSC curves of c) LLG, d) CMLLG.

two proton peaks at 4.6 and 4.2 ppm that could be attributed to methylene proton in carboxymethyl substituents [11,45]. Fig. 5a and b depicts the NMR of LLG and CMLLG, respectively. 3.2.3.5. Elemental analysis. The carbon, hydrogen and oxygen content of LLG were found to be 48.60%, 6.57% and 37.54%, respectively. After carboxymethylation the content of carbon, hydrogen and oxygen in CMLLG was increased, i.e. 51.10%, 7.01% and 40.51%, respectively, as compared to LLG which was due to the attachment of carboxymethyl groups by replacing hydroxyl groups of LLG [30]. 3.2.3.6. Zeta potential. Zeta potential of LLG was found to be −2.58 mV and for CMLLG the value obtained was −9.80 mV. The results revealed more negative potential in the case of CMLLG as compared to LLG indicating the more negatively charged moiety (carboxyl group) onto the galactomannan structure which suggests its utility for modulating drug release characteristics through gum–polymer or gum–ion interactions. 3.2.3.7. SEM. The morphological investigations of the LLG and CMLLG was done by scanning electron microscopy and results revealed that the LLG shows discrete, elongated, irregular granular structure (Fig. 4c). The granules of LLG have an irregular but smooth surface and no defects were seen on the surface of gran-

ules [43]. On the other hand CMLLG structure (Fig. 4d) showed lots of small pores, which resulted in surface roughness. The alkaline environment of reaction, during the carboxymethylation process could be responsible for these structural changes [43,47]. Further, the crystallinity of LLG also seems to be altered in SEM images of CMLLG. These results are consonance with the results revealed in DSC and XRD studies.

3.2.3.8. Rheological study of LLG and CMLLG. The rheological properties of LLG and CMLLG were examined at different concentrations. Fig. 6a and b represents the viscosity curves measured at different concentrations ranging from 1.0 to 7.0% (w/v) of LLG and CMLLG, respectively. The galactomannan solutions were displaying low viscosity at lower concentrations (1–2% w/v). The concentrated solutions (3–7% w/v) were not only exhibiting higher viscosities, also the rate of viscosity increase in these concentrations was faster. The greater viscosities at higher concentrations were attributed to an enhanced entanglement of galactomannan chains [40]. In addition, the strengthening of the topological constraints posed to each individual chain by the polymer network and greater intermolecular forces (hydrogen bonding) due to an increased number of hydroxyl groups at high concentrations also increases the viscosity [48]. The solutions were found to exhibit shear thinning behaviour at increasing shear rate due to decrease in chain entanglements.

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Fig. 4. XRD diffractogram of a) LLG, b) CMLLG and SEM images of c) LLG, d) CMLLG.

Fig. 5.

1

H NMR spectra of a) LLG and b) CMLLG.

Similar behavior, i.e. non-Newtonian pseudoplastic has also been reported by Verma and Razdan [49]. Further, this shear thinning behaviour with increasing shear rate was more evident in higher concentrations as compared to lower concentrations. CMLLG solutions exhibited higher viscosities (1.5-3 times) than native gum. The introduction of carboxymethyl groups in LLG increases the intramolecular repulsion due to the presence of similar charges.

This repulsion leads to an extended conformation of CMLLG resulting in an increased viscosity [50]. 3.2.3.9. Swelling index of LLG and CMLLG in distilled water and in different media. The CMLLG displayed a higher swelling (11.0 ± 3.1 to 17.8 ± 1.6) in distilled water as compared to LLG (8.0 ± 1.1). The introduction of carboxymethyl groups in galactomannan increases

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Fig. 6. Flow curves of a) LLG and b) CMLLG solutions as a function of rotation rate at different concentrations.

Fig. 7. Schematic representation depicting relationship between pH and swelling index of a) LLG and b) CMLLG.

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the hydrophilicity of gum thus increasing the swelling index of gum. Further, the water solubility of CMLLG increases with an increase in substitution [11]. The batch with the highest DS was selected (CMLLG11, DS = 0.805) for swelling studies in different media. LLG was found to hydrate quickly and swell in both acidic (pH 1.2) and basic media (pH 8.0), because it is little affected by pH or ionic strength due to its neutral nature, therefore swelling index of LLG was same in different media (Fig. 7a). There was no statistical significant difference between swelling of LLG (p < 0.05) in both acidic and basic buffers. A pH dependent swelling was observed in the case of CMLLG which was due to the presence of carboxymethyl groups. The carboxymethyl groups of CMLLG ionize at a pH above the pKa of carboxylic groups (3.4–3.7) and the deprotonation of carboxylic groups at pH 6.8 and 8.0 results in electrostatic repulsion forces between ionized acid groups, thus higher swelling index was observed (Fig. 7b). The CMLLG displayed a low swelling index at pH 1.2. At this pH the carboxyl groups are protonated and promote the formation of intramoleculer hydrogen bonding. Similar results have been reported in polymers containing pendant carboxylic acid groups such as carboxymethyl-k-carrageenan and locust bean gum [51].

3.2.4. Mechanical properties Firmness designates maximum force required to extrude the sample from concentric annular space between the plunger and the container. Higher firmness is exhibited by samples possessing higher viscosities which were attributed to a decrease in work of shear. An increase in concentration resulted in an increase in

consistency values. The greater value of cohesiveness and index of viscosity was observed with CMLLG than LLG due to greater viscosity. Fig. 8 depicts the mechanical properties of the LLG and CMLLG. These texture properties (firmness, consistency, cohesiveness and index of viscosity) are important indicators as viscosifying agents in food and pharmaceuticals.

4. Conclusion The water soluble hydrocolloid was extracted by aqueous method, identified and its different properties were characterized. The extracted gum was found to be galactomannan with nonNewtonian pseudoplastic (shear thinning) behavior. The extracted galactomannan was successfully derivatized and the reaction parameters were optimized.The maximum degree of substitution of the derivative was found to be 0.805 with good hydrophilic and swelling properties. The excellent functional properties of the gum indicated that it can be used as stabilizer, emulsifier and suspending agent. All these properties of LLG and CMLLG suggest that galactomannan possess sufficient potential for use in the pharmaceutical industry.

Acknowledgement The authors would like to acknowledge the financial assistance provided by the Department of Science and Technology (DST, SERB), New Delhi (DST No. SB/FT/LS-177/2012, Dated 26/04/2013).

Fig. 8. Mechanical properties of LLG and CMLLG at different concentrations.

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References [1] R.L. Whistler, Introduction to Industrial Gums, R.L. Whistler J.N. Be Miller (Eds.), Industrial gums: Polysaccharides and their derivatives, New York, 1990, pp.5–7. [2] H. Bhatia, P.K. Gupta, P.L. Soni, Int. J. Sci. Environ. Tech. 2 (2013) 708–724. [3] J.S.G. Reid, Adv. Bot. Res. 11 (1985) 125–155. [4] G. Robinson, S.B. Ross-Murphy, E.R. Morris, Carbohydr. Res. 107 (1982) 17–32. [5] V.C. Pandey, A. Kumar, Genet. Resour. Crop Evol. 60 (2013) 1165–1171. [6] S. Rastogi, U.N. Dwivedi, Regeneration and transformation of tree legumes with special reference to Leucaena species, in: P.K. Jaiwal, R.P. Singh (Eds.), Focus on Biotechnology: Applied Genetics of Leguminosae Biotechnology, Kluwer Academic Publishers, Dordrecht, 2003, pp. 329–359. [7] A.R. Chowdhury, R. Banerji, G. Misra, S.K. Nigam, J. Am. Oil Chem. Soc. 61 (1984) 1023–1024. [8] H.P. Ramesh, K. Yamaki, T. Tsushida, Carbohydr. Polym. 50 (2002) 79–83. [9] D. Syamsudin, R. Sumarny, P. Simanjuntak, Eur. J. Sci. Res. 43 (2010) 384–391. [10] M.S. Buckeridge, S.M.C. Dietrich, D.U. Lima, Galactomannans as the reserve carbohydrate in legume seeds, in: A.K. Gupta, N. Kaur (Eds.), Carbohydrate Reserves in Plants-synthesis and Regulation, Elsevier, Netherlands, 2000, pp. 283–316. [11] G. Dodi, D. Hritcu, M.I. Popa, Cellul. Chem. Technol. 45 (2011) 171–176. [12] K.S. Parvathy, N.S. Susheelamma, R.N. Tharanathan, A.K. Gaonkar, Carbohydr. Polym. 62 (2005) 137–141. [13] R. Malviya, P. Srivastava, M. Bansal, P.K. Sharma, Asian J. Pharm. Clin. Res. 3 (2010) 238–241. [14] F.O. Ohwoavworhua, T.A. Adelakun, Trop. J. Pharm. Res. 4 (2007) 501–507. [15] H.A. Pawar, K.G. Lalitha, Int. J. Biol. Macromol. 65 (2014) 167–175. [16] M. Jindal, V. Kumar, V. Rana, A.K. Tiwary, Carbohydr. Polym. 93 (2013) 386–394. [17] D.R. Picout, S.B. Ross-Murphy, Carbohydr. Polym. 70 (2007) 145–148. [18] L.M. Nwokocha, P.A. Williams, Carbohydr. Polym. 90 (2012) 833–838. [19] R. Singh, R. Malviya, P.K. Sharma, Pharmacogn. J. 3 (2011) 17–19. [20] P.A. Dakia, C. Blecker, C. Robert, B. Wathelet, M. Paquot, Food Hydrocolloids 22 (2008) 807–818. [21] G. Kaur, D. Singh, V. Brar, Int. J. Biol. Macromol. 70 (2014) 408–419. [22] H. Goel, G. Kaur, A.K. Tiwary, V. Rana, Yakugaku Zasshi. 130 (2010) 729–735. [23] P.R. Rai, A.K. Tiwary, V. Rana, Carbohydr. Polym. 87 (2012) 1098–1104. [24] F. Grzegorzewski, R. Sascha, L.W. Kroh, M. Geyer, O. Schluter, Food Chem. 122 (2010) 1145–1152.

841

[25] R.W. Eyler, E.D. Klug, F. Diephuis, Anal. Chem. 19 (1947) 24–27. [26] Y.L. Lopez-Franco, C.I. Cervantes-Montano, K.G. Martínez-Robinson, J. Lizardi-Mendoza, L.E. Robles-Ozuna, Food Hydrocolloids 30 (2013) 656–660. [27] R. Sharma, M. Ahuja, Carbohydr. Polym. 85 (2011) 658–663. [28] A. Kumar, M. Ahuja, Carbohydr. Polym. 90 (2012) 637–643. [29] H.S. Mahajan, V.K. Tyagi, R.R. Patil, S.B. Dusunge, Carbohydr. Polym. 91 (2013) 618–625. [30] S. Kaity, A. Ghosh, Ind. Eng. Chem. Res. 52 (2013) 1033–1045. [31] A. Haddarah, A. Bassal, A. Ismail, C. Gaiani, I. Ioannou, C. Charbonnel, T. Hamieh, M. Ghoul, J. Food Eng. 120 (2014) 204–214. [32] K. Edsman, J. Carlfors, R. Petersson, Eur. J. Pharm. Sci. 6 (1998) 105–112. [33] M. Jindal, V. Kumar, V. Rana, A.K. Tiwary, Food Hydrocolloids 30 (2013) 192–199. [34] A.M. Unrau, J. Org. Chem. 26 (1961) 3097–3101. [35] V.M. Devi, V.N. Ariharan, N.P. Prasad, Res. J. Pharm. Biol. Chem. Sci. 4 (2013) 515–521. [36] A.K. Singh, R.P. Selvam, T. Sivakumar, Int. J. Biomed. Res. 1 (2010) 35–41. [37] V.D. Prajapati, G.K. Jani, N.G. Moradiya, P.N. Randeri, J.B. Nagar, N.N. Naikwadi, B.C. Variya, Int. J. Biol. Macromol. 60 (2013) 83–92. [38] A. Lafuma, D. Quere, Nat. Mater. 2 (2003) 457–460. [39] N. Mittal, G. Kaur, J. Appl. Polym. Sci. 131 (2014) 1–9. [40] H. Gong, M. Liu, J. Chen, F. Han, C. Gao, B. Zhang, Carbohydr. Polym. 88 (2012) 1015–1022. [41] P. Goyal, V. Kumar, P. Sharma, P. Carbohydr Polym. 69 (2007) 251–255. [42] O.S. Lawal, M.D. Lechner, W.M. Kulicke, Polym. Degrad. Stab. 93 (2008) 1520–1528. [43] L.F. Wang, S.Y. Pan, H. Hu, W.H. Miao, X.Y. Xu, Carbohydr. Polym. 80 (2010) 174–179. [44] Y. Huang, H. Yu, C. Xiao, Carbohydr. Polym. 66 (2006) 500–513. [45] P.J. Manna, T. Mitra, N. Pramanik, V. Kavitha, A. Gnanamani, P.P. Kundu, Int. J. Biol. Macromol. 75 (2015) 437–446. [46] P. Bassi, G. Kaur, Eur. J. Pharm. Biopharm. 96 (2015) 173–184. [47] J.H. Trivedi, K. Kalia, N.K. Patel, H.C. Trivedi, Carbohydr. Polym. 60 (2005) 117–125. [48] R. Fijan, S. Sostar-Turk, R. Lapasin, Carbohydr. Polym. 68 (2007) 708–717. [49] P.R.P. Verma, B. Razdan, J. Sci. Ind. Res. 61 (2002) 437–443. [50] S.U. Sajjan, M.R. Rao, J. Sci. Food Agric. 48 (1989) 377–380. [51] Z. Mohamadnia, M.J. Zohuriaan-Mehr, K. Kabiri, A. Jamshidi, H. Mobedi, J. Bioact. Compat. Polym. 22 (2007) 342–356.