Utilization of waste dried Mangifera indica leaves for extraction of mangiferin by conventional batch extraction and advance three-phase partitioning

2.1 Material

Leaves of M. indica were obtained from the Institute of Chemical Technology garden at Matunga, Mumbai, India. The leaves were washed and sun dried for 48 h. The dried leaves were then powdered and stored in an airtight container in a cool place. The moisture content of leaves was 9%, and the powder size was about 1–2 mm. The mangiferin used as the reference standard (Sigma M3547-100 mg ≥98% HPLC grade) was purchased from Sigma-Aldrich (USA). All solvents were of analytical grade and purchased from Hi Media Ltd. (Mumbai, India). Methanol and deionized acidified water (0.1% acetic acid) were used as the mobile phase. Ammonium sulfate was procured from S. D. Fine Chemicals Limited (Mumbai, India). All experiments were performed three times, and average values are reported with standard deviations.

2.2 Analysis of mangiferin

Analysis of mangiferin was carried out by HPLC (Agilent 1260 infinity high-performance auto sampler). All experiments were performed on a C18 column. The analysis conditions were maintained by isocratic elution with a flow rate of 1 ml/min. The mobile phase used was methanol and acidified water in 30:70 v/v ratio. Samples were analyzed using HPLC. The peak areas at 254 nm were measured. All experiments were performed three times to check the reproducibility, and their average values are reported. Statistical analysis was done using one-way ANOVA, and p values were obtained. The values were considered statistically significant if the p values were <0.05.

2.3 Preparation of TPP

TPP involves adding a salt and organic solvent simultaneously to the slurry. This mixture is allowed to mix well, which results in the formation of three phases. It is believed that many factors like kosmotropy, conformation tightening and electrostatic forces play a role in the TPP process [16]. Slurry was prepared by dissolving 0.5 g of M. indica leaves powder in 20 ml of distilled water. Ammonium sulfate (3.4 g, 30% w/v) was added gradually along with intermediate stirring in 250 ml reactor containing 20 ml of slurry. This was followed by the addition of 20 ml of t-butanol. This mixture was kept in a glass reactor equipped with a stirrer at 500 rpm for 60 min. The temperature was maintained at 30±2°C for all the experiments. Later, the mixture was allowed to stand at 30±2°C for 1 h to form three phases. The organic and aqueous layers were collected, and the t-butanol was evaporated on a rotary vacuum evaporator. Analysis of both layers was done by HPLC. In order to get the optimum extraction yield by TPP, parameters affecting the TPP process were studied. Parameters such as variation in the salt, salt concentration, the ratio of slurry to t-butanol, stirring time, pH and solute to solvent ratio were optimized.

2.4 Soxhlet extraction

Soxhlet extraction was used as a reference method for comparison with the TPP. Soxhlet extraction was carried out for 5 h using water as a solvent.

2.5 Batch extraction

Batch extraction was performed in a glass reactor of 250 ml capacity equipped with a six-bladed (pitched blade) glass turbine for agitation. Mangifera indica leaves powder (0.5 g) was put in a glass reactor, and 40 ml water was added. The temperature of experiments was maintained at 30±2°C. The mixture was then agitated for 3 h 40 min at a speed of 500 rpm. Samples (0.1 ml) were withdrawn at regular intervals of 20 min and then diluted and centrifuged for analysis using HPLC.

Several parameters affecting the extraction of mangiferin from M. indica leaves in a batch process were studied. Parameters such as stirring time, solute to solvent extraction, stirring speed and temperature were optimized.

3.1 Three-phase partitioning

3.1.1 Effect of screening of salts on TPP of mangiferin

Earlier work on TPP used ammonium sulfate salt to precipitate proteins [16]. At higher salt concentrations, the surface charges are neutralized resulting in precipitation of the proteins. Different salts will precipitate out different proteins or biomolecules. The ionic strength of the solution will influence the solubility of proteins or any other biomolecules. Various salts like ammonium sulfate, magnesium sulfate and sodium sulfate were used to study its effect on TPP. These salts were selected because sulfate ions are among the early members of the Hofmeister series. A solution of each salt at 1 m was used by keeping parameters like pH 6, slurry to t-butanol ratio 1:1, solute to solvent ratio 1:40, temperature 30±2°C and ammonium sulfate concentration 30% w/v constant for stirring time 60 min. The results obtained are depicted in Figure 1, which show that ammonium sulfate gave the maximum extraction yield. This might be due to the fact that ammonium sulfate is more water soluble as compared to the other two salts. Also, the sulfate ions act as a dehydrating agent, thus increasing their effective radius. Thereby, large ions crowd together, thus segregating proteins out of the water phase [19]. Thus, it can be concluded that the dehydrating action of sulfate ions alters the solubility of mangiferin creating unfavorable conditions for mangiferin in the aqueous phase and thereby pushing mangiferin into the organic phase. Further, by keeping previously mentioned experimental parameters like pH 6, slurry to t-butanol ratio 1:1, solute to solvent ratio 1:40 and temperature 30±2°C constant, ammonium sulfate was varied from 30% to 60% w/v concentration for a stirring time of 60 min. As can be seen in Figure 2, the yield of mangiferin (21 mg/g) was highest at 40% w/v. Thus, 40% w/v ammonium sulfate concentration is selected as the optimum concentration for a further set of experiments.

Figure 1:

Effect of screening of salts on TPP of mangiferin at pH 6, slurry to t-butanol ratio 1:1, solute to solvent ratio 1:40, temperature 30±2°C and ammonium sulfate concentration 30% w/v constant.

Figure 2:

Effect of ammonium sulfate concentration on TPP of mangiferin at pH 6, slurry to t-butanol ratio 1:1, solute to solvent ratio 1:40, temperature 30±2°C constant.

3.1.2 Effect of time on TPP of mangiferin

The time required for extraction is critical in determining the cost of the process, and thus it is essential to obtain the optimum time of extraction. Experiments were performed separately for different stirring times ranging from 30 min to 150 min keeping the process parameters constant such as pH 6, slurry to t-butanol ratio 1:1, solute to solvent ratio 1:40, temperature 30±2°C and 40% w/v ammonium sulfate concentration. Figure 3 showed that there was an increase in the extraction of mangiferin from 30 min to 120 min, and yield remained constant after 150 min of stirring. This is due to saturation of the solvent beyond 150 min. Therefore, extraction time was fixed at 120 min for the subsequent experiments.

Figure 3:

Effect of stirring time on TPP of mangiferin at pH 6, slurry to t-butanol ratio 1:1, solute to solvent ratio 1:40, temperature 30±2°C and 40% w/v ammonium sulfate concentration.

3.1.3 Effect of slurry to t-butanol ratio on TPP of mangiferin

The effect of slurry to t-butanol ratio on the extraction of mangiferin was studied by varying the ratio of slurry to t-butanol as 1:0.5, 1:1, 1:2, 1:4 and 1:8 at pH 6, solute to solvent ratio 1:40, stirring time 2 h, temperature 30±2°C and ammonium sulfate concentration 40% w/v. It was observed that extraction of mangiferin was highest at a 1:1 ratio as shown in Figure 4. Tertiary butanol acts as a kosmotrope agent in synergism with sulfate. A lesser quantity of t-butanol does not provide an adequate synergistic effect with ammonium sulfate [16]. Hence, a lesser extraction yield is seen at a ratio of 1:0.5. Further, increase in the t-butanol volume beyond 1:1 ratio leads to a decrease in concentration gradient between both the phases, resulting in a decreased mass transfer. Thus, the extraction yield of mangiferin decreases. Hence, 1:1 slurry to t-butanol ratio was considered optimum for a further set of experiments [17].

Figure 4:

Effect of slurry to t-butanol ratio on TPP of mangiferin at pH 6, solute to solvent ratio 1:40, stirring time 2 h, temperature 30±2°C and ammonium sulfate concentration 40% w/v.

3.1.4 Effect of pH on TPP of mangiferin

The effect of pH on the extraction process using HCl and NaOH was analyzed. The pH was varied from 5 to 9 by keeping other parameters constant like the ratio of slurry to t-butanol 1:1, solute to solvent ratio 1:40, stirring time 2 h, temperature 30±2°C and ammonium sulfate concentration 40% w/v. As per Figure 5, mangiferin extraction was significantly higher in the acidic range as compared to the basic range. At acidic pH, solubility of ammonium sulfate in water is very high compared to mangiferin. Thus, the solubility of mangiferin is altered in an aqueous phase as large sulfate ions become crowded in an aqueous phase creating unfavorable conditions for mangiferin. Hence, mangiferin is pushed in t-butanol phase, and higher extraction in t-butanol is achieved.

Figure 5:

Effect of pH on TPP of mangiferin at solvent ratio 1:40, stirring time 2 h, temperature 30±2°C and ammonium sulfate concentration 40% w/v.

3.1.5 Effect of solute to solvent ratio on TPP of mangiferin

The effect of solute to solvent ratio, i.e. amount of mangiferin powder to solvent (water) was studied by varying it at 1:20, 1:30, 1:40 and 1:50 at pH 6, slurry to t-butanol 1:1, stirring time 2 h, temperature 30±2°C and ammonium sulfate concentration 40% w/v. From the results shown in Figure 6, it is observed that the maximum yield of 28 mg/g was obtained at ratios of 1:40 and 1:50. Consequently, 1:40 was selected as an optimum ratio. The reason for obtaining higher yields at 1:40 and 1:50 than the 1:20 and 1:30 ratio could be related to the fact that at lower solute to solvent ratio there is less concentration gradient leading to less mass transfer and less reduction in yield. However, varying solute to solvent ratio did not increase the yield further due to the equilibrium. Thus, it is essential to maintain a proper balance of solute to solvent ratio for getting optimum yield.

Figure 6:

Effect of solute to solvent ratio on TPP of mangiferin at pH 6, slurry to t-butanol 1:1, solute to solvent ratio 1:40, stirring time 2 h, temperature 30±2°C and ammonium sulfate concentration 40% w/v.

3.2 Batch extraction

3.2.1 Effect of stirring time on batch extraction of mangiferin

The time of stirring was optimized from 0 min to 3 h 40 min. The effect of stirring on yield was monitored and shown in Figure 7 at solute to solvent ratio 1:40, stirring speed 400 rpm and temperature 30±2°C. It was observed that from 20 min, the yield of mangiferin increased gradually up to 160 min and then remained constant up to 220 min. The maximum yield of 16 mg/g was obtained at 160 min. The initial increase in the concentration of mangiferin is due to the large concentration difference between extracting solvent and the mangiferin present on the surface of a cell wall from which the mangiferin molecules diffuses into the solvent. As extraction continues, the concentration difference decreases leading to a decrease in the extraction rate. Also, after 160 min, available mangiferin present on the surface of a cell wall is not available for diffusion, and hence the extraction yield remains almost constant up to 220 min. Hence, all the experiments were performed for 160 min.

Figure 7:

Effect of stirring time on batch extraction of mangiferin at solute to solvent ratio 1:40, stirring speed 400 rpm and temperature 30°C.

3.2.2 Effect of solute to solvent ratio on batch extraction of mangiferin

The effect of solute to solvent ratio, i.e. amount of mangiferin powder to solvent (water) on batch extraction was studied to avert the undue use of the solvent. Solute to solvent ratio was varied as 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 by keeping other parameters like stirring speed 400 rpm and temperature 30±2°C constant. It is observed that the maximum yield was obtained at ratios of 1:40 and 1:50 (Figure 8). Extraction yield increased as the ratio decreased from 1:20 to 1:40, after which it remained constant. This is due to the addition of the higher amount of solvent providing the larger concentration gradient which favors the mass transfer. Thus, 1:40 was considered to be the optimum ratio for the extraction of mangiferin.

Figure 8:

Effect of solute to solvent ratio on batch extraction of mangiferin at stirring speed 400 rpm, time 160 min and temperature 30°C.

3.2.3 Effect of stirring speed on batch extraction of mangiferin

In extraction, the energy is provided in the form of agitation making it essential to optimize the stirring speed. Experiments are carried out from 200 to 500 rpm for 160 min at solute to solvent ratio 1:40 and temperature 30±2°C. From Figure 9, it can be seen that the yield increases when speed increases from 200 to 400 rpm due to reduction in mass transfer resistance, but beyond that no change has been observed. This is attributed to fact that the extremal mass resistance is negligible at 400 rpm and further agitation will not provide any improvement in mass transfer. Thus, 400 rpm speed is sufficient for extraction of mangiferin; hence, all further experiments are carried out at 400 rpm speed.

Figure 9:

Effect of stirring speed on batch extraction of mangiferin at solute to solvent ratio 1:40, time 160 min and temperature 30°C.

3.2.4 Effect of temperature on batch extraction of mangiferin

With an increase in temperature, the diffusivity of mangiferin in water increases, and it was expected that the extraction would also increase. Thus, extraction was carried out at different bath temperatures, i.e. 30°C, 40°C, 50°C, 60°C and 70°C for 160 min at solute to solvent ratio of 1:40 and stirring speed 400 rpm, and results are shown in Figure 10. When the temperature is increased from 30°C to 50°C, extraction yield increases. It remains almost constant beyond 50°C till 70°C. Increase in the temperature of solvent increases the solubility of mangiferin. Similarly, the viscosity of the solvent reduces at higher temperature which results in a higher diffusion of the solvent through the pores. However, marginal change in extraction yield from 50°C to 70°C suggests that 60°C is the suitable temperature for extraction of mangiferin.

Figure 10:

Effect of temperature on batch extraction of mangiferin at solute to solvent ratio 1:40, time 160 min and stirring speed 400 rpm.

3.3 Comparison of Soxhlet, batch extraction and TPP

The extraction yields obtained at optimized parameters for TPP and batch extraction were compared with Soxhlet extraction. The mangiferin yield of 28 mg/g was obtained in TPP in 120 min, while batch extraction yielded 23 mg/g in 160 min. There is a considerable reduction in time requirement in TPP compared to the batch extraction. Although the time required using TPP has reduced by 40 min, there is no significant change in the extraction yield as compared with batch extraction. The yield obtained by Soxhlet extraction was 57 mg/g; however, the time required was 5 h. Thus, simple TPP is not suitable to extend the extraction of mangiferin, and batch-stirred extraction can be used for the large-scale extraction due to its low cost.