Synthesis, physico-chemical and biomedical applications of sulfated Aegle marmelos gum: Green chemistry approach

2.1 Materials

Bael fruits, unripe were collected from the local market. De-ionized (Milli-Q) water was used for all experiments. Sulfuric acid was purchased from the Himedia Laboratories, Mumbai, India. All other chemicals used were of analytical reagent grade.

2.2 Antinutritional factors

Dried mesocarp and pericarp from unripe bael fruits was tested for the presence of antinutritional factors. The extracted gum was tested particularly for those antinutritional factors that could be expected to be present in dried mesocarp and pericarp.

2.3 Extraction of BFG

BFG was extracted by modifying the method reported by Jindal et al. (2013). Briefly, partially ripe bael fruits were collected from the A. marmelos tree. The hard woody and spherical fruits were carefully broken down into two equal parts. The amber colored viscous, very sticky, translucent gummy substance along with the seeds and pulp separating the fruit outer wall was marked as the desired portion. This gum along with seeds was collected in a beaker containing 2% v/v glacial acetic acid solution. The slurry was boiled on a water bath for 45 min with continuous stirring and kept overnight. The slurry was filtered through a muslin cloth to remove the debris. The gum was precipitated from the filtered slurry by adding acetone. The precipitates were dried in a vacuum oven at 50 °C and grounded to obtain a light brown fine powder. The gum was further purified by dialysis and purified BFG was obtained by freeze drying.

2.4 Modification of BFG

The effect of three factors, the concentration of sulfuric acid, reaction time and reaction temperature on the degree of sulfation was investigated. Nine reacting conditions were designed according to orthogonal test as L₉ (3³) (Table 1). Three levels per factor were employed using 9.5, 19 or 38 M concentration of sulfuric acid, reaction time of 2, 4 or 6 h, and reaction temperature of 0, 10 or 25 °C, respectively.

2.5 Ultrasonication of BFG

200 mg of BFG was suspended in sulfuric acid (9.5, 19 or 38 M) before being subjected to ultrasonication for 2, 4 or 6 h at 0, 10 or 25 °C. The samples were subsequently neutralized using 0.1 N NaOH, dialyzed and eventually lyophilized. The results represent average values of three experiments.

2.6 Elemental analysis of BFG

Lyophilized BFG samples were analyzed for sulfur content (% S) by elemental analysis using ElementarVarioMicro (Elementar, Analysensysteme, Germany). Degree of sulfation (DS) was calculated according to the following equation (Bae et al., 2009): $$\mathit{DS}=\frac{0.162\times (\%S/32)}{100-[(80/32)\times \%S}$$ where, % S is the sulfur content (%).

2.7 Swelling index

The BFG or sulfated uBFG1 samples (100 mg) were soaked in distilled water, HCl (0.1 N) or phosphate buffer of pH 1.2, 6.8 or 7.4, respectively (100 cc) for 24 h. The swollen material was then removed and weighed after superficial drying using a blotting paper. The swelling index (SI) was calculated as: $$\mathit{SI}=\frac{\mathit{wf}-\mathit{wi}}{\mathit{wi}}$$ where, w_f is the weight of swollen material and w_i is the initial weight of the dry material.

2.8 Zeta potential studies

The zeta potentials of BFG and uBFG1 were measured at 25 °C by using Zetasizer 4 (Malvern Instrument Ltd., UK). Samples were diluted with HPLC water (MilliQ Synergy Systems, Millipore) and placed in a capillary measurement cell.

2.9 FTIR-ATR analysis

FTIR-ATR spectra of BFG and uBFG1 were recorded on a FTIR-ATR spectrophotometer (Alfa, Bruker, Berlin, Germany). The FTIR-ATR spectra were obtained between wavelengths of 4000 and 400 cm⁻¹.

2.10 Rheological behavior

2.11 Mechanical properties

Back extrusion (BE) and cone penetration (CP) tests were performed for investigating the rheological behavior. Both experiments were performed using a TA XT Plus Texture Analyzer (Stable Micro Systems Ltd, Godalming, UK) equipped with a 300N load cell.

A rig (model A/BE, Stable Micro Systems) consisting of a flat 35 mm diameter perspex disc plunger that was driven into a larger perspex cylinder sample holder (50 mm diameter) to force down into the sample was used for BE measurements. The movement of the plunger forced the sample to flow upward through the concentric annular space between the plunger and the container. The measuring cup was filled with 30 ± 1 g of BFG, uBFG1 or SA sample. The test was replicated eight times at a pretest speed of 1.0 mm s⁻¹, test speed of 2.0 m s⁻¹ at a distance of 50 mm above the top of the sample, penetrated to a depth of 10 mm, and returned to the starting position. At this point (most likely to be the maximum force), the probe returned to its original position. The maximum positive force of extrusion (firmness [N]), area of the curve (consistency [N.s]), and maximum negative force due to back extrusion (cohesiveness [N]) and the negative area of extrusion (index of viscosity [N·s]) were documented as descriptors of rheological behavior of these samples.

Spreadability rig (HDP/SR, Stable Micro Systems) was used for CP determination. It consisted of a 45° conical perspex probe (P/45C) that penetrated a conical sample holder containing BFG, uBFG1 or SA sample. Product was penetrated to a distance of 50 mm at a 3 mm/s compression rate. The work required to accomplish penetration was calculated from the area under the curve (work of shear [N.s]).

2.12 Antimicrobial effect

The antimicrobial activities of BFG, uBFG1 and SA samples against Bacillus cereus and E. coli were examined (Jindal et al., 2013). B. cereus and E. coli were inoculated in nutrient broth and incubated at 37 °C for 24 h. The gum solutions (90 μL) with three different concentrations (0.5, 1.0, 2.0 mg/mL) were added to the culture broth (10 μL) which was incubated at 37 °C for 18 h, and then the absorbance of the culture broth was measured at 540 nm. The microbial inhibition effect was calculated as follows; $$\text{Inhibition effect}(\%)=[(\text{Abs of control}-\text{Abs of sample})/\text{Abs of control}]\times 100$$

2.13 Anticoagulant activity

The anticoagulant activity of BFG, uBFG1 or SA sample was determined by using the method of Matsubara et al. (2001). For the activated partial thromboplastin time (APTT) assay, uBFG1 sample (concentration of 25, 50 or 100 μg/mL prepared in 50 μL in distilled water) was mixed with the plasma (50 μL) and incubated at 37 °C for 2 min. Then, APTT assay reagent (100 μL) was added to the resulting solution and further incubated at 37 °C for 6 min. After the addition of 20 mM CaCl₂ (100 μL), the clotting time was recorded and compared with that of heparin (Himedia laboratories Ltd., Mumbai, India). In the prothrombin time (PT) assay, PT assay reagent (100 μL) preincubated at 37 °C for 10 min was added to the solution of the sulfated derivative (50 μL) and plasma (50 μL) and the clotting time was measured. The prothrombin time (International Normalized Ratio) was obtained from the clotting time ratio between the sample and control.

3.1 Antinutritional factors

The results obtained, showed the absence of hemagglutinating activity, saponins, trypsin inhibitors and α-amylase inhibitors in the dried mesocarp and pericarp of the bael fruit. The absence of these antinutritional factors is known to improve the nutritional value of the bael fruit and consequently of BFG. Alkaloids were found to be present in the dried mesocarp and pericarp of the bael fruit, whereas, BFG was devoid of alkaloids.

1.07 mg g⁻¹ of phytic acid was found to be present in the dried mesocarp and pericarp of the bael fruit. Phytate is considered an antinutritional factor mainly due to its ability to bind essential dietary minerals, proteins and starch, which often resulted in reduced absorption of these important constituents. However, several researchers have contested that phytic acid has antioxidant and anticancer properties (Vucenik and Shamsuddin, 2006). In addition, it is used in the preparation of various food products such as bread, pasta and meat products. The bioavailability of proteins or minerals is also reported to be reduced by tannins (Ferreira et al., 2004). Although, BFG was found to contain 1.21 mg of tannic acid per 100 mg⁻¹, no phytic acid or tannins was detected in BFG. Absence of amylase inhibitors, trypsin inhibitors, and lack of hemagglutinating activity improves the safety and the nutritional quality of BFG.

3.2 Sulfation of BFG

A. marmelos gum is highly branched in nature with terminal units of galactose, galactutronic acid, rhamnose and arabinose (Roiy et al., 1977). The present investigations revealed the content of galacturonic acid in BFG to be approximately 7%. The ratio of the sugar terminal units to sugars in the chains is 1:2. The gum contains 1 → 3 linkages in the galactose backbone where galactose sugar hydroxyls at positions 2, 4 and 6 are free. Further, in the terminal hexose sugar unit, four hydroxyls at position 1, 3, 4 and 6 are free. Hence, both free hydroxyls present in galactose as well as in the hexose units can be envisaged to be easily amenable to sulfation. Sulfation of BFG was attempted under different conditions that were obtained by varying the reaction parameters in order to obtain the maximum DS. The reaction parameters were varied with respect to the concentration of sulfuric acid, temperature and time (Table 1). The results indicated the highest yield of 149 mg of uBFG1 while the lowest yield was 15 mg of uBFG9. The DS of uBFG followed the order: uBFG1 > uBFG2 > uBFG3 > uBFG5 > uBFG4 > uBFG6 > uBFG8 > uBFG7 > uBFG9 and were 1.94, 1.48, 1.06, 0.91, 0.74, 0.68, 0.29, 0.17 and 0.15, respectively. Analysis of the orthogonal array design indicated that the most predominant influence on DS was exerted by variable A (molar ratio of sulfuric acid). The extent of the impact of variables on DS followed the order: variable C (reaction time) < B (reaction temperature) < A (molar ratio of sulfuric acid). It was observed that maximum DS was obtained using 9.5 M of sulfuric acid with a reaction time of 2 h at 0 °C. The extent of sulfation for all samples was observed to be relatively low as compared to that reported earlier. Previous studies have reported the sulfation of curdlan by conventional chemical methods to range from 5.6% to a maximum of 20% (Yoshida et al., 1995). However, these conventional chemical processes utilize pyridine-SO₃ complex that generates harmful waste (pyridine) and also require a long refluxing period as well as a multi-step preparation.

3.3 Swelling index

The swelling of BFG in different media was observed to follow the order pH 7.4 = pH 6.8 > pH 1.2 > HCl (0.1 N) (Fig. 1). The uBFG1 sample also exhibited the same pattern (Fig. 1). However, the SI of uBFG1 was higher than that of BFG in any buffer. It is well established that low swelling in acidic pH restricts the release of drugs from dosage forms. At the same time, high swelling in alkaline pH would be useful for sustaining drug release for a prolonged period as the dosage form travels down the g.i.t (Rai et al., 2012). Therefore, higher magnitude of swelling of uBFG1 as compared to BFG can be expected to be useful for modulating the drug release from dosage forms.

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Figure 1 Swelling indices of BFG and uBFG1 in various pH enviornments.

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3.4 FTIR analysis

Fig. 2 depicts the ATR spectra of BFG and uBFG1 samples. In the spectrum of BFG (Fig. 2a), the absorption bands at 1613 and 1421 cm⁻¹ could be ascribed to asymmetrical and symmetrical COO⁻ stretching vibrations, respectively. The disappearance of these two absorption bands in the spectra (Fig. 2b) of uBFG1 and appearance of a new band at 1734 cm⁻¹ suggested C⚌O stretching vibration. Furthermore, the disappearance of this band on treatment of uBFG1 with dilute NaOH solution indicated that it to be due to C⚌O stretching vibration of carboxylic acid moieties present in uBFG1 sample. Fig. 2b revealed additional two bands each at 1258 and 854 cm⁻¹. These bands could be ascribed to asymmetrical S⚌O stretching vibration and symmetrical C–O–S vibration possibly associated to a C–O–SO₃ group, respectively. Additionally, the band at 1636 cm⁻¹ could be related to the unsaturated bond formed due to the sulfation process. These data have been shown in Table 2.

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Figure 2 FTIR-ATR spectra of (a) BFG and (b) uBFG1.

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3.5 Zeta potential

The zeta potentials of BFG and uBFG1 were found to be −16.7 and −38.95 mV, respectively, which indicate the presence of anionic groups. Moreover, aqueous dispersions of BFG and uBFG1 yielded an acidic pH, as is evident from FTIR that BFG and uBFG1 have uronic acid and sulfate groups, respectively. The results of negative zeta potential are in consonance with FTIR results. This behavior of BFG and uBFG1 is similar to that observed for other polysaccharides and was attributed to the presence of uronic acid and sulfate units in their structure, respectively. The high negative zeta potential of both the samples suggests their utility in enforcing gum-polymer or gum–ion interactions for modulating drug release characteristics. The higher zeta potential of uBFG1 sample could be attributed to greater the electronegative character of ${\mathit{SO}}_3^{2-}$ groups, which would exhibit a higher magnitude of crosslinking thus yielding greater cross-linked density.

3.6 Rheological behavior

The viscosity of both BFG and uBFG1 samples was observed to increase with an increase in their concentrations. However, the increase in viscosity of uBFG1 samples was much greater than for BFG samples. Further, uBFG1 exhibited significantly greater (p < 0.05) viscosity as compared to BFG dispersions of comparable concentration (Fig. 3a). Accordingly the force required to accomplish penetration of a probe to a fixed depth (work of shear) was observed to be greater for uBFG1 samples (Fig. 3b). The SI of the uBFG1 sample was greater than that of the BFG solution in both the acidic as well as alkaline pH (Fig. 1). The higher SI of the uBFG1 sample is supported by its higher zeta potential, due to which, greater quantity of water molecules would have got associated with it.

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Figure 3 Comparison of viscosity and textural behavior of BFG, uBFG1 and SA; (a) viscosity (b) work of shear.

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3.7 Mechanical properties

Neither BFG sample nor sodium alginate (SA) sample was found to exhibit any significant (p < 0.05) change in the firmness (Fig. 4a) and cohesiveness (Fig. 4b) values with increasing concentrations. Also, the consistency (Fig. 4c) and index of viscosity (Fig. 4d) were not markedly affected by changing their concentration. However, the firmness (Fig. 4a), cohesiveness (Fig. 4b), consistency (Fig. 4c) and index of viscosity (Fig. 4d) of the uBFG1 sample increased with an increase in concentration.

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Figure 4 Comparison of viscosity and mechanical properties of BFG, uBFG1 and SA; (a) firmness (b) consistency (c) cohesiveness and (d) index of viscosity.

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Firmness is a measure of the maximum force required to extrude a sample from concentric annular space between the plunger and container. Hence, it is logical to expect greater firmness for samples of uBFG1 that had exhibited greater viscosities at different concentrations (Fig. 3a) as compared to both SA and BFG samples. The fact was that uBFG1 samples showed greater firmness, the area under the curve obtained was also greater. It reflected higher consistency values with increasing concentrations. Similarly, the less viscous SA and BFG samples did not exhibit any marked change in the maximum force due to back extrusion (cohesiveness). Whereas, the samples of uBFG1, that exhibited greater viscosities with increasing concentrations, revealed greater cohesiveness and thereby, greater index of viscosity.

SA has been widely investigated for its use in modifying drug release dosage forms and food preparations (Jensen et al., 2012) due to its viscous behavior. A critical appraisal of the results revealed that the viscosity of SA samples was less than those of BFG and uBFG1 samples. Parameters obtained from texture analysis evidently indicated that firmness, cohesiveness, consistency and index of viscosity of SA was comparable to BFG. uBFG1 was significantly (p < 0.05) superior to both SA and BFG with respect to these parameters. The fact that these parameters are important indicators of the performance of viscosifying agents in food and pharmaceuticals, uBFG1 could be suggested to offer a great potential over the widely used SA for use in pharmaceutical dosage forms and food products.

3.8 Antimicrobial effect

Antimicrobial effects of BFG, uBFG1 and SA samples were evaluated using B. cereus and E. coli as a model system. Overall, the uBFG1 exhibited better antimicrobial effects than BFG and SA at all concentrations tested for B. cereus (Fig. 5a). Even, BFG significantly (p < 0.05) inhibited the growth of B. cereus and E. coli by 17% and 27% at a concentration of 2.0 mg/mL, respectively (Fig. 5a and b). It implies that the sulfation enhanced the antimicrobial activity of gum. In addition, the antimicrobial effect had a tendency to increase with increasing concentration of the sulfated derivative. It was previously reported that the bacteriostatic activity of carrageenans was critically dependent on the presence of sulfate groups (Yamashita et al., 2001). Cellulose sulfate also exhibited antimicrobial activity against Neisseria gonorrhoeae and Chlamydia trachomatis, which was explained by the interaction between proteoglycan receptors and their target cell ligands (Anderson et al., 2002). Moreover, uBFG1 was observed to be more effective against E. coli as compared to B. cereus. Thus, the sulfation-derived antimicrobial activity appeared to be more effective against E. coli which belongs to the gram-negative bacteria. Similar observations have been reported by Park et al. (1995) who found the pectin hydrolysate to be more effective against gram-negative bacteria (E. coli and A. aceti) than gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis).

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Figure 5 Antimicrobial activities of BFG, uBFG1 and SA against; (a) B. cereus and (b) E. coli. n = 3 for each group; Values are mean ± S.D. Data of the results were analyzed using ANOVA followed by the Tukey’s multiple range test. a = P < 0.05 vs. BFG 0.5 mg/ml; a^∗ = P < 0.05 vs. SA 0.5 mg/ml. b = P < 0.05 vs. BFG 1.0 mg/ml; b^∗ = P < 0.05 vs. SA 1.0 mg/ml. c = P < 0.05 vs. BFG 2.0 mg/ml; c^∗ = P < 0.05 vs. SA 2.0 mg/ml.

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3.9 Anticoagulant activity

Blood coagulation is a complex process involving the sequential activation of clotting factors, ultimately leading to the formation of insoluble fibrin. The disorders of coagulation can give rise to an increased risk of bleeding and/or clotting. Since heparin has been clinically used as an effective anticoagulant medicine, the anticoagulant activity of the sulfated gum (uBFG1) was compared with that of heparin. Fig. 6 summarizes the anticoagulant activity of sulfated gum sample. The sulfated gum derivative significantly (p < 0.05) prolonged prothrombin time (PT) in a concentration-dependent manner. It is evident that incorporation of sulfate groups into the gum structure was responsible for the anticoagulant activity since native gum hardly exhibited any anticoagulant effect. The APTT (activated partial thromboplastin time) for uBFG1 was recorded to be more than 4 min at a concentration of 25–100 μg/mL. This could be attributed to the anionic character of the sulfated gum, which would have made it easy to interact with positively charged coagulation proteins, thus, improving the anticoagulant activity. Studies have demonstrated that chemical sulfation of pectins, including citrus pectin, give products with anticoagulant properties (Bae et al., 2009), with the activity depending on the quantity of sulfate groups. Anticoagulant effects have been reported for other sulfated polymers such as β-glucan, chitosan, galactan and galactomannan (Bae et al., 2009). Therefore, the results confirmed the enhanced anticoagulation activity of uBFG1 to be due to the introduction of sulfate groups in BFG. The prolonged PT and APTT (Fig. 6) indicated the inhibition of extrinsic and intrinsic coagulation pathways, respectively.

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Figure 6 Comparison of anticoagulant activity of heparin with BFG, uBFG1 and SA n = 3 for each group; Values are mean ± S.D. Data of the results were analyzed using ANOVA followed by the Tukey’s multiple range test. a = P < 0.05 vs. heparin 1.0 μg/ml; a^∗ = P < 0.05 vs. BFG 25 μg/ml; a^∗∗ = P < 0.05 vs. SA 25 μg/ml. a = P < 0.05 vs. heparin 5.0 μg/ml; a^∗ = P < 0.05 vs. BFG 50 μg/ml; a^∗∗ = P < 0.05 vs. SA 50 μg/ml. a = P < 0.05 vs. heparin 10.0 μg/ml; a^∗ = P < 0.05 vs. BFG 100 μg/ml; a^∗∗ = P < 0.05 vs. SA 100 μg/ml.

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