ENHANCING THE EXTRACTION OF POLYPHENOLS FROM ACACIA LEUCOPHLOEA BARK BY MICROWAVE-ASSISTED ENZYMATIC EXTRACTION METHOD AND IN-VITRO STUDY OF ITS ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY

ENHANCING THE EXTRACTION OF POLYPHENOLS FROM ACACIA LEUCOPHLOEA BARK BY MICROWAVE-ASSISTED ENZYMATIC EXTRACTION METHOD AND IN-VITRO STUDY OF ITS ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY

Sudha Ayyasamy ^* and Sangeetha Velusamy

Department of Food Technology, Kongu Engineering College, Perundurai, Erode - 638060, Tamil Nadu, India.

ABSTRACT: This study aims to identify the suitable method for extraction of polyphenols from Acacia leucophloea bark using enzymes and to optimize the processing condition for extraction using Response Surface Methodology. A microwave-assisted enzymatic extraction (MAEE) method was developed and optimized to augment the polyphenol yield. The optimal extraction conditions were as follows: the amount of cellulase 1%, agitation speed 108 rpm, incubation time 1.5 h, and irradiation time 6 min. Under these conditions, polyphenol yield could reach 6.31%, which was higher than other extraction methods studied. The antioxidant activity of the crude extract and the encapsulated extract was evaluated by DPPH radical and reducing power assay. The antibacterial activities of crude extract and the encapsulated extract were studied using the disc diffusion technique. Among the tested bacteria, Klebsiella pneumoniae was most susceptible to polyphenol extract with the highest inhibition zone of 18.6 ± 0.5 mm at the concentration of 8 mg/mL. The outcome of the study indicates that the MAEE method was efficient and eco-friendly, and the polyphenols have remarkable antioxidant and antibacterial activities.

Microwave-assisted enzymatic extraction, Polyphenols, Acacia leucophloea, Antioxidant activity, Antimicrobial activity, medicinal plant

INTRODUCTION: Free radicals and reactive oxygen species (ROS) are generated in the body as byproducts of cellular metabolism. It was reported that free radicals and ROS might cause oxidative damage to living cells, which leads to the development of chronic diseases such as aging, heart diseases, stroke, arthritis, and cancers. Our body has a natural defense mechanism consisting of glutathione peroxidase, catalase, and superoxide dismutase against free radicals.

However, these endogenous antioxidants are not able to combat efficiently and therefore, supply of antioxidants through diet is required to reduce the oxidative damage due to ROS in our system ^{1, 2}.

Antioxidants are not only needed to diminish ROS in the body but also needed for use as food additives. Thus more attention has been focussed toward finding natural antioxidants, which could clear free radicals efficiently. Antioxidants act by inhibiting the initiation and propagation steps and thereby delaying the oxidation process. They also act as scavengers of free radicals ³. Antioxidants used as food additives can be either natural or synthetic. The toxicity and carcinogenic activity of synthetic antioxidants direct the research towards natural and safer antioxidants for food applications ⁴.

Plant sources are rich in natural antioxidants such as tocopherol, vitamin C, carotenoids, phenolic acids, and flavonoids ². Natural antioxidants have anti-viral, anti-inflammatory, anti-cancer, anti-mutagenic, anti-tumour, and hepatoprotective properties ^{1, 5}. The change in consumer demand for naturally processed, additive-free, and safe products has occurred ⁶. So, the replacement of synthetic antioxidants by natural ones interests food technologists.

Acacia leucophloea (Roxb.) Willd. (Syn. Mimosa leucophloea) (Mimosoideae), native to south and southeast Asia, is a non-contiguous large thorny tree attaining height of 35 m and diameters at breast height of 100 cm. It is distributed in arid India, Sri Lanka, Bangladesh, Burma, and much of Thailand ². It has a white to yellowish-gray bark, which exfoliates in long strips. The bark of the plant has been used in Indian traditional medicine.

It is used in the treatment of fever, dysentery, gum bleeding, diabetes, dry cough, mouth ulcers, skin diseases, and ulcers, as it has antimicrobial, anthel-mintic, expectorant, and blood purifying activity. The gum and bark decoction is used as an anti contraceptive and menstrual complaint regulator. The chemical constituents found in the tree are n-hexacosanol, beta-amyrin, beta-sitosterol, and tannins ⁷. The extracts from the bark were found to have phenolics and flavanoids ⁸.

Enzyme-assisted extraction can be used to increase bioactive components release from plant material, which reduces extraction time and usage of solvents and provide increased yield and quality of the product. Various enzymes such as cellulases, pectinases, and hemicellulase are often utilized to disrupt the structural integrity of the plant cell wall, thereby enhancing the extraction of bioactives from plants ⁹. These enzymes have been studied to be efficient for extraction of polyphenols from plant sources such as kinnow peel, orange peel, Grape pomace, Pinus taiwanensis and apple pulp ^10-14.

Microwave-assisted enzymatic extraction (MAEE) is a novel and green extraction technique that can offer high reproducibility in a shorter time, simplified manipulation, reduced solvent consumption, and lower energy input without decreasing the extraction yield of the target species ¹⁵. As a new-type extraction technique, MAEE is known as a more environmental-friendly process with economic advantages than the traditional extraction methods. In recent times, this technique has been commonly used for sample preparation ¹⁶. There is limited work on the study of polyphenol extraction and antioxidant activity of Acacia leucophloea bark.

Therefore, the objective of this work is to study the feasibility of microwave-assisted enzymatic extraction (MAEE) for the polyphenols from Acacia leucophloea bark and to evaluate their antioxidant and antibacterial activities in-vitro. Box-Behnken design (BBD) combined with response surface methodology (RSM) was applied to study the interaction among MAEE operating parameters. Moreover, the effect of micro-encapsulation on antioxidant and antibacterial activities of Acacia leucophloea bark extract from MAEE was also studied.

MATERIALS AND METHODS:

Plant Material and Reagents: The bark of Acacia leucophloea was collected from different places of Tirupur and Erode districts, Tamil Nadu, India. Freshly collected bark of Acacia leucophloea was dried at 40℃ in a tray dryer (CM Envirosystems-Humidry-TD-12-S-E) and pulverized using a ball mill (Everflow) to a coarse powder and passed through 1.4 mm mesh sieve.

2,2-diphenyl-1-picrylhydrazyl (DPPH) and tert-Butylhydroquinone (TBHQ) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Gallic acid was obtained from Himedia, India, and cellulase was obtained from Sisco Research Laboratory Pvt., Ltd., Mumbai, India. All other chemicals used in the study were of analytical grade.

Methods of Extraction:

Non-Enzymatic Extraction: Powder of 1.0 g dried Acacia leucophloea bark was weighed and added into a round bottom flask with 50 mL 70% (v/v) ethanol. The flask was placed in a heating mantle and connected with a condenser, and then allowed to reflux for 3.0 h at 65 °C.

Enzymatic Extraction: Powder of 1.0 g dried Acacia leucophloea bark was weighed and added into a conical flask. To the flask, 50 mL of the enzymatic solution was prepared by dispersing 1.0 wt. % (with respect to solid sample) cellulase in distilled water was added, and then pH was adjusted to 5.5 using 0.1N HCl or 0.1N NaOH solution. The flask was kept in an orbital shaker (Remi CIS-24BL) and then allowed to incubate at 55°C and agitated at 100 rpm for 2 h.

Microwave-Assisted Enzymatic Extraction (MAEE): Powder of 1.0 g dried Acacia leucophloea bark was weighed and added into a conical flask with 25 mL double distilled water. The flask was sealed and microwave irradiated at 160W for a defined time (4.0-8.0 min). After microwave irradiation, an enzymatic solution prepared by dispersing (0.5-1.5 wt. % with respect to solid sample) cellulase in distilled water was added to the samples, and then pH was adjusted to 5.5 using 0.1N HCl or 0.1N NaOH solution. The flask was kept in an orbital shaker (Remi CIS-24BL) and then allowed to incubate at 55 °C and agitated at a different speed (80-120 rpm) for 1.0-2.0 h. The residues were then filtered, and total polyphenols in the filtrates were determined. All experiments were conducted in triplicate.

Concentration of the Extract: The filtrate was concentrated by drying in tray dryer at 50 °C and stored until needed for the bioassays at -4 °C.

Experimental Design: A four-variable, three-level Box–Behnken design (BBD) was applied to determine the best combination of extraction variables for the polyphenol yield from the bark of Acacia leucophloea. The four independent variables were the amount of cellulase (%, X₁), agitation speed (rpm, X₂), incubation time (h, X₃), and irradiation time (min, X₄), and each variable was set at three levels. The uncoded (actual) levels of the independent variables are given in Table 1.

TABLE 1: CODE AND LEVEL OF FACTORS CHOSEN FOR THE STUDY

Twenty-nine experiments, augmented at the center points five times, were carried out. Regression analysis was performed for the experiment data and was fitted into the empirical second-order polynomial model, as shown in the following equation:

Where β_o is the offset term, β_i is the i^th linear coefficient, β_ii is the quadratic coefficient, β_ij is the ij^th interaction coefficient, X_i and X_j are input variables, real values converted from coded values, which influence the response variable Y.

Determination of Total Polyphenolic Content: The total polyphenols content of the extract was obtained by following the Folin-Ciocalteau method ¹⁷. 0.1 mL aliquots of dilute extracts were adjusted to 3 mL by adding distilled water and shaken with 0.5 mL of Folin-Ciocalteau reagent. After 3 min, 2 mL of 20% (w/v) sodium carbonate was added, and the mixture was shaken well and kept at 50 °C for 1 min. After incubation, the absorbance was measured at 650 nm on a spectrophotometer (Elico SL244). The results are expressed as a percentage of polyphenol content (gallic acid equivalents) in the raw material studied.

Antioxidant Activities:

DPPH Radical Scavenging Activity Assay: The DPPH radical-scavenging assay was used to determine the antioxidant activity of the polyphenols extract following the method of Zhang et al. ¹⁸ The 0.2 mmol/L solution of DPPH in methanol was freshly prepared before use. Serial dilutions of the extracts were carried out. Solutions (2 mL each) were then mixed vigorously with methanolic solution DPPH (2 mL) and allowed to stand in the dark for 30 min at ambient temperature for any reaction to occur, and the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity, which was analyzed from the graph plotted of inhibition percentage against compound concentration. TBHQ (tert-butyl hydroquinone) was used as a positive control. All tests were performed in triplicate and averaged. The scavenging activity was estimated based on the percentage of DPPH radical scavenged as the following equation:

Where Ac is the absorbance of DPPH solution without sample, Ai is the absorbance of the sample with DPPH solution, and Aj is the absorbance of the sample without DPPH solution.

Reducing Power: The reducing power of the extract was determined according to the method of Vaquero et al. ¹⁹ Extract aliquots of varying concentration was mixed with phosphate buffer (0.2 M, pH 6.6) and 1% (w/v) potassium ferricyanide. After the mixture was incubated at 50 ºC for 25 min, 10% (w/v) trichloroacetic acid was added, and the mixture was centrifuged at 3000 rpm for 10 min. The upper layer of solution was mixed with distilled water and 0.1% ferric chloride for 10 min, and then the absorbance was measured at 700 nm against a blank. Increasing absorbance of the reaction mixture indicates increasing reducing power ¹⁸. TBHQ was used as a positive control. All tests were performed in triplicate and averaged.

Encapsulation of the Extract: As a large number of polyphenolic compounds show limited water solubility and unpleasant taste, it insists the formulation of a finished protecting product, which is able to maintain the structural integrity, mask its taste, increase its water solubility and bioavail-ability, and convey it precisely towards a physio-logical target. Among the existing stabilization methods, encapsulation is an interesting means. The extract obtained from the MAEE method at optimized conditions was then mixed with 10% maltodextrin, 0.3% tween 80, and mixed using a beater until foam was retained. The foam obtained was scattered on the pan and dried at 60 °C for 2h in tray dryer (CM Envirosystems-Humidry-TD-12-S-E). The dried powder was blended and sifted using a sieve 100 mesh ²⁰.

Antibacterial Activity:

Microbial Strains and Inoculum Preparation: The in-vitro antibacterial activity of the polyphenol extract was evaluated against four pathogenic microorganisms viz. Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris and Staphylococcus aureus. All strains stock cultures were maintained at 4 °C on nutrient agar medium. Active cultures were prepared by inoculating fresh nutrient broth medium with a loopful of cells from the stock cultures at 37 °C for overnight. To get desirable cell counts for bioassays, overnight grown bacterial cells were subcultured in fresh nutrient broth at 37 °C ²¹.

Disc Diffusion Method: The antimicrobial activity of extract of Acacia leucophloea bark was screened using the disc diffusion technique. Inoculum of 0.1 mL from microorganism suspensions (10⁶ CFU/ml) was spread on the solid agar plates and was allowed to stand for 15 min. Autoclaved filter paper discs of 6mm were loaded with 250 μL of polyphenol extracts of different concentrations (2, 4, 6, 8 mg/mL). Cefotaxime (1 mg/mL) was used as a positive control to determine the sensitivity of bacterial species tested. The loaded disc was placed on the surface of the medium and incubated at 37 °C for 24 h. The diameters of the inhibition zones were measured in millimeters. These studies were performed in triplicate.

Determination of MIC by Visual Analysis: The minimal inhibitory concentration (MIC) of poly-phenol extract was determined by serial dilution technique ²². The tested concentrations of poly-phenol extract ranged from 0.025 to 5 mg/mL ¹⁸.

Statistical Analysis: All results of twenty nine experiments in Box-Behnken design were analyzed using Design Expert® software version 8.0.7.1 from Statease Inc., Minneapolis. The significance of the intercept, linear, squared, and interaction coefficients of the factors was evaluated by analysis of variance (ANOVA).

RESULTS AND DISCUSSION:

Comparison of MAEE with other Extraction Methods: Polysaccharides are the major barrier to the release of polyphenolic compounds. A recent study has revealed that the formation of the complex polysaccharide–phenol entities might be due to two mechanisms of association, viz., (1) hydrogen bonds and (2) hydrophobic interactions²³. Cellulase can catalyze the breakdown of the rigid skeleton of cellulose and degrade the plant cell wall constituents to improve the release of polyphenolic compounds ²⁴. Microwave-assisted extraction (MAE) is a fast extraction process where microwave energy is delivered efficiently to materials through molecular interaction with the electromagnetic field and offers a rapid transfer of energy to the extraction solvent and raw plant materials ^{25, 26}. Furthermore, the direct interaction of microwave with solvent also results in the rupture of the plant cells and release of intracellular products into the solvent quickly ^{27, 28}. As both cellulase and MAE are independent cases and are effective in rupturing the cell, they must be combined together to check whether the enzyme and MAE can work together to improve polyphenol extraction yield.

The efficiency of extracting polyphenols from Acacia leucophloea bark by different methods, including non-enzymatic extraction, enzymatic extraction, and microwave-assisted enzymatic extraction (MAEE) was studied. The extraction conditions and yields are shown in Table 2.

TABLE 2: COMPARISON OF POLYPHENOL YIELD BY DIFFERENT EXTRACTION METHODS

^a The values of polyphenol yield are expressed as mean and standard deviation (SD) calculated from three independent experiments.

^bPolyphenol yield is a percentage of extracted polyphenol content in the raw material studied

Table 2 shows that non-enzymatic extraction yield (about 1.9 ± 0.1%) was inferior compared with those of enzymatic extraction and MAEE. It can be also established that the extraction yield (5.7 ± 0.16%) obtained by MAEE was higher than that (4.3 ± 0.15%) by enzymatic extraction. The highest polyphenol yield using MAEE indicated that MAEE was more efficient than the other two methods. This higher efficiency could be recognized as microwave irradiation, which produces the disruptions of tissues and cell walls, leading to a greater contact area between solid and liquid phase, better access of solvent to valuable components ²⁹.

Optimization of MAEE Conditions using Box–Behnken Design:

Fitting the Model:

TABLE 3: BOX-BEHNKEN DESIGN WITH EXPERIMENTAL RESPONSES

TABLE 4: ESTIMATED REGRESSION COEFFICIENTS AND ANALYSIS OF VARIANCE (ANOVA) FOR THE QUADRATIC POLYNOMIAL MODEL

Table 3 presents the experiment design and corresponding response data for the polyphenols yield from Acacia leucophloea bark. The regression coefficients of the intercept, linear, quadratic, and interaction terms of the model were are shown in Table 4. It was evident that all linear and all the interaction parameters were significant at the level of P < 0.01, whereas one quadratic parameter was insignificant (P > 0.05). Neglecting the non-significant parameter, the final predictive equation obtained was as follows:

Y = - 49.9263 + 12.856 X₁ + 0.484042 X₂ + 12.66567 X₃ + 4.625167 X₄ -0.065 X₁ X₂ +1.37 X₁ X₃ - 0.01825 X₂ X₃ - 0.00863 X₂ X₄ - 0.36 X₃ X₄ - 3.43633 X₁² - 0.00158 X₂² - 3.18133X₃² - 0.27633 X₄²                                                                                                             (3)

The analysis of variance (ANOVA) for the experimental results is given in Table 4. For the model fitted, the determination coefficient (R²) was 0.996, which implied that the sample variations of 99.6% for the MAEE efficiency of Acacia leucophloea bark polyphenols were attributed to the independent variables, and only 0.4% of the total variations cannot be explained by the model. F-value for the lack of fit was insignificant (P > 0.05), thereby confirming the validity of the model. The value of the coefficient of variation (C.V.) was 1.2%, suggesting that the model was reliable and reproducible ^{30, 31}. The results indicated that the model could work well for the prediction of polyphenols extract from Acacia leucophloea bark.

Analysis of Response Surfaces: In order to investigate the interactive effects of operational parameters on polyphenols extraction, the three-dimensional response surface plots were generated by plotting the response on the Z-axis against two independent variables while keeping the other two independent variables at their middle value.

Phenols are more linked to the cell wall and are contained in vacuoles, and several factors can affect phenols released from cell walls and vacuoles, including the amount of enzyme. The amount of enzyme is a critical variable, which can improve the extraction of phenols from the cell wall. Apparently, the enzyme assisting the release of phenols from the cell-wall matrix occurs via an enzyme catalysed hydrolytic degradation of the cell wall polysaccharides and breakage of the ether and/or ester linkages between the phenols and the plant cell-wall polymers ²³. However, it must be taken into account that the amount of enzyme is applied for extraction with two main aims: (1) to calculate the amount of enzyme that is required for complete extraction, and (2) to avoid the excess use of enzymes ²⁴.

FIG. 1: RESPONSE SURFACE PLOTS SHOWING EFFECTS OF THE EXTRACTION PARAMETERS ON POLYPHENOL YIELD: (A) AT VARYING AMOUNT OF CELLULASE (%) AND AGITATION SPEED (RPM), (B) AT VARYING AMOUNT OF CELLULASE (%) AND INCUBATION TIME (H) (C) AT VARYING AMOUNT OF CELLULASE (%) AND IRRADIATION TIME (MIN) (D) AT VARYING AGITATION SPEED (RPM) AND INCUBATION TIME (H) (E) AT VARYING AGITATION SPEED (RPM) AND IRRADIATION TIME (MIN) (F) AT VARYING INCUBATION TIME (H) AND IRRADIATION TIME (MIN)

Fig. 1a-c depicts the interactions between the amount of cellulase and each of the three other factors (agitation speed, incubation time, and irradiation time) on the extraction yield of polyphenols. As can be seen from Fig. 1a-c that the extraction yield of polyphenols increased gradually with increasing amounts of cellulase from 0.5 to 1.2 wt.%, while increasing the amount of cellulase from 1.2% to 1.5%, the polyphenol yield was maintained at 6.2 %, indicating that 1.2% enzyme provided sufficient amounts of activity for the hydrolysis of the cell-wall.

The interactions of agitation speed with each of the three other factors on the extraction yield of polyphenols are shown in Fig. 1a, d, and e. In all three cases, the extraction yield increased rapidly with increasing agitation speed from 80 to 107 rpm, and then followed by a decline with the further increase. A similar influence of the agitation speed has been observed in various studies. Excessively high mixing speeds (200 rpm) lowered the extent of cellulose conversion, while moderate mixing speeds (100-200 rpm) provided a good combination of fast initial hydrolysis rates and high conversion yields in Avicel and paper pulp ³². Cellulose hydrolysis by cellulase enzymes requires adequate mixing to ensure sufficient contact between the substrate and enzymes and to promote heat and mass transfer within the reaction vessel. Some agitation increases the hydrolysis rate and yields, but excessive mixing can deactivate the enzymes and reduce the conversion yield ^{33, 34}.

The deactivation effect has been attributed to the shear force generated by the mixer and the entrapment of air bubbles into the medium at the air-liquid surface ³⁵.

Fig. 1b, d, and f display the interactions between incubation time and each of the three other factors on the polyphenols extraction yield. The results showed that the polyphenols extraction yield increased with the extension of incubation time till 1.6 h.

The interaction of irradiation time with each of the three other operational parameters on the polyphenol yield is shown in Fig. 1c, e, and f. It can be observed from the figure that the polyphenol yield increased with increasing irradiation time and reached a maximum value in 6 min. After that, longer irradiation time caused negative effects due to degradation or conversion of the polyphenols¹⁸.

Verification of the Predictive Model: By carrying out parameter optimization on the basis of the built mathematical models, the obtained experimental conditions for polyphenol yield were: the amount of cellulase 1.00wt%, agitation speed 108 rpm, incubation time 1.54 h, and irradiation time 6.28 min. Under the optimal parameters, the predictive value of polyphenol yield was 6.28%. Because the values of incubation time and irradiation time are difficult to operate in the actual extraction experiments, they were carried out with slight modifications: incubation time in 1.5 h and irradiation time in 6 min. The experimental polyphenol yield of 6.31% at optimized conditions was consistent with the predicted value. The strong correlation between the real and predicted results confirmed that the response model was satisfactory to reflect the expected optimization condition.

Antioxidant Activity: Phenolic compounds have exhibited strong antioxidant activities and are able to quickly reduce reactive oxygen species (ROS), including free radicals, thereby protecting biomolecules (e.g., polyunsaturated fatty acids) against oxidation ³⁶. In this study, the antioxidant activities of the polyphenol extracts from Acacia leucophloea bark were evaluated by using in vitro antioxidant models, namely DPPH radical scavenging assay and reducing power.

DPPH Radical Scavenging Assay: The hydrogen atom or electron donation abilities of some pure compounds were measured by bleaching a purple-colored methanol solution of the stable DPPH radical. The DPPH solution has a deep violet colour, and radical scavenging activity of antioxidant compounds by the loss of absorbance as the pale yellow non-radical form (DPPH-H) is produced ².

FIG. 2: ANTIOXIDANT ACTIVITIES OF THE POLYPHENOL EXTRACT FROM ACACIA LEUCOPHLOEA BARK BY MAEE METHOD: (A) DPPH RADICAL SCAVENGING ACTIVITY (B) REDUCING POWER. CE, CRUDE EXTRACT; TBHQ, TERT-BUTYLHYDROQUINONE (MEAN ± D, N=3)

Fig. 2a exhibits the radical scavenging capacities of the polyphenol extracts on DPPH (TBHQ as positive control). From Fig. 2a, a dose-response relationship was observed in the DPPH radical scavenging activity. The activity increased with increasing concentration of the polyphenol extract. Crude extract has shown DPPH radical scavenging activity of 80.33% higher than TBHQ (77.66%) in a concentration of 0.1mg/mL and lemon peel essential oil which showed 54.67% scavenging ability ³⁷. These results indicated that the scavenging effect of polyphenol extract on DPPH radical was remarkable.

Reducing power: In the reducing power assay, the presence of reductants (antioxidants) in the samples would result in the reduction of the Fe³⁺/ ferricyanide complex to its ferrous form (Fe²⁺) by donating an electron. Hence, the Fe²⁺ can then be monitored by measuring the formation of Perl’s Prussian blue at 700 nm ³⁸. A higher absorbance value indicates higher reducing power.

Fig. 2b presented the reductive capabilities of polyphenol extracts and compared with TBHQ as control standards. As seen in Fig. 2b, the reducing power of all the samples increased gradually with the increase in concentration tested. The reducing power of the crude extract was found to be lower than TBHQ in the tested concentration range. The reducing power may increase with the purified extract of Acacia leucophloea bark, as shown by purified extract of polyphenols from peanut shells ¹⁸.

Encapsulated Acacia leucophloea Bark: Natural polyphenols are valuable compounds possessing scavenging properties towards radical oxygen species and complexing properties towards proteins. Unfortunately, these properties are also responsible for a lack of long-term stability. Moreover, poly-phenols often present a poor biodisponibility mainly due to low water solubility. Lastly, many of these molecules possess a very astringent and bitter taste, which limits their use in food or in oral medications. To circumvent these drawbacks, delivery systems have been developed, and among them, encapsulation would appear to be a promising approach ³⁹.

The extract of Acacia leucophloea bark obtained at optimized conditions of MAEE method was encapsulated and its total polyphenol content, scavenging activity by DPPH assay, and reducing power were found to be 3.6 %, 59.19%, and 0.49 (1mg powder/mL). Desai and Park noted that maltodextrin could improve the stability of phenol compounds as maltodextrin can protect phenol compounds from oxidation effect, oxygen, water, and extreme temperature ⁴⁰.

Antibacterial Activity: The results of antibacterial activity against four bacterial species are shown in Table 5. It can be observed that crude extract possessed an inhibitory effect on the bacterial species studied (Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus). The inhibitory zones for the bacterial strains were in the range of 8.3 ± 0.5 to 10.3 ± 0.5 (2 mg/mL), 10.3 ± 0.2 to 12.1 ± 0.2 (4 mg/mL), 12.5 ± 0.5 to 15.0 ± 0.5 (6 mg/mL) and 13.8 ± 0.7 to 18.6 ± 0.5 (8 mg/mL), respectively. The zone of inhibition was increased with increasing the concentration of polyphenol extract. The composition and concentration of secondary metabolites determine the antimicrobial efficacy of plants. The bark extracts of Acacia leucophloea were found to have steroids, polyphenols, tannins, alkaloids, gums, and mucilages, which could be responsible for the medicinal properties of this plant ⁴¹. Among the tested bacteria, Klebsiella pneumoniae was most susceptible to polyphenol extract with the highest inhibition zone of 18.6 ± 0.5 mm at the concentration of 8 mg/mL ⁴².

On the other hand, low antibacterial activity was exhibited against Pseudomonas aeruginosa and Staphylococcus aureus (14.8 ± 0.7 and 13.8 ± 0.7 mm, respectively). The encapsulated extract had been found to demonstrate remarkable antibacterial activity against the tested bacterial species. In addition, the minimal inhibitory concentration (MIC) of polyphenol extract was found in the 0.5-1.0 mg/mL concentration range. The lowest MIC value (0.5 mg/mL) was obtained for Klebsiella pneumoniae.

TABLE 5: ANTIBACTERIAL ACTIVITIES OF CRUDE EXTRACT (CE) ZONE OF INHIBITION AND MINIMUM INHIBITORY CONCENTRATIONS (MIC)

^a Diameter of inhibition zones (mm) including the diameter of the disc (6 mm), values are expressed as the mean ± SD (n=3).

^b Minimal inhibitory concentration (mg/mL) of CE.

CONCLUSION: In the present study, the extraction yield of total polyphenols using MAEE method was higher than other extraction methods. The operational parameters for enhanced extraction of polyphenols from Acacia leucophloea bark by MAEE process were optimized with a Box-Behnken design based on response surface methodology. The optimal extraction conditions of MAEE using response surface methodology obtained were as follows: the amount of cellulase 1%, agitation speed 108 rpm, incubation time 1.5 h, and irradiation time 6 min.

The predicted polyphenol yield was well consistent with the experimental value. The bark extract and encapsulated product exhibited DPPH radical scavenging activity, reducing power, and antimicrobial activity. The results of the present study may be helpful to further exploit and utilize this resource.

ACKNOWLEDGEMENT: The authors are thankful to the management of Kongu Engineering College, Perundurai, Tamilnadu, India, for providing the necessary facilities to carry out the research work.

CONFLICTS OF INTEREST: None

REFERENCES:

1. Lim YY and Murtijaya J: Antioxidant properties of Phyllanthus amarus extracts as affected by different drying methods. LWT-Food Science and Technology 2007; 40: 1664-69.
2. Gulcin I, Bursal E, Sehitoglu MH, Bilsel M and Goren AC: Polyphenol contents and antioxidant activity of lyophilized aqueous extract of propolis from Erzurum, Turkey. Food and Chemical Toxicol 2010; 48: 2227-38.
3. Gulcin I: Antioxidant activity of caffeic acid (3, 4-dihydroxycinnamic acid). Toxicology 2006; 217: 213-20.
4. Gulcin I: Antioxidant and antiradical activities of L-carnitine. Life Sciences 2006; 78: 803-11.
5. Mihaylova D, Vrancheva R, Desseva I, Ivanov I, Dincheva I, Popova M and Popova A: Analysis of the GC-MS of volatile compounds and the phytochemical profile and antioxidant activities of some Bulgarian medicinal plants. Zeitschrift für Naturforschung C 2018; 74: 45-54.
6. Bianco A and Uccella N: Biophenolic components of olives. Food Research International 2000; 33: 475-85.
7. Murugan K, Senthilkumar B, Senbagam D and Al-Sohaibani S: Biosynthesis of silver nanoparticles using Acacia leucophloea extract and their antibacterial activity. International Journal of Nanomedicine 2014; 9: 2431-38.
8. Katalinic V, Mozina SS, Generalic I, Skroza D, Ljubenkov I and Klancnik A: Phenolic profile, antioxidant capacity, and antimicrobial activity of leaf extracts from six Vitis vinifera varieties. International Journal of Food Properties 2013; 16: 45-60.
9. Puri M, Sharma D and Barrow CJ: Enzyme-assisted extraction of bioactives from plants. Trends in Biotechnology 2012; 30: 37-44.
10. Puri M, Kaur A, Schwarz WH, Singh S and Kennedy JF: Molecular characterization and enzymatic hydrolysis of naringin extracted from kinnow peel waste. International Journal of Biological Macromolecules 2011; 48: 58-62.
11. Li BB, Smith B and Hossain MM: Extraction of phenolics from citrus peels: II. Enzyme-assisted extraction method. Separation and Purification Technology 2006; 48: 189-96.
12. Kammerer D, Claus A, Schieber A and Carle: A novel process for the recovery of polyphenols from grape (Vitis vinifera) pomace. Journal of Food Science 2005; 70: C157-C163.
13. Lin SC, Chang CMJ and Deng TS: Enzymatic hot pressurized fluids extraction of polyphenolics from Pinus taiwanensis and Pinus morrisonicola. Journal of the Taiwan Institute of Chemical engineers 2009; 40: 136-42.
14. Missang CE, Massiot P, Baron A and Drilleau JF: Effect of oxidative browning of apple pulp on the chemical and enzymatic extraction of cell wall polysaccharides. Carbohydrate Polymers 1993; 20: 131-38.
15. Xu DP, Li Y, Meng X, Zhou T, Zhou Y, Zheng J, Zhang JJ and Li HB: Natural antioxidants in foods and medicinal plants: extraction, assessment and resources. International Journal of Molecular Sciences 2017; 18: 96-127.
16. Chen Y, Xie MY and Gong XF: Microwave-assisted extraction used for the isolation of total triterpenoid saponins from Ganoderma atrum. Journal of Food Engineering 2007; 81: 162-70.
17. Singleton VL and Rossi JL: Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. The American Journal of Enology and Viticulture 1965; 16: 144-58.
18. Zhang G, Hu M, He L, Fu, P, Wang L and Zhou J: Optimization of microwave-assisted enzymatic extraction of polyphenols from waste peanut shells and evaluation of its antioxidant and antibacterial activities in-vitro. Food and Bioproducts Processing 2013; 91: 158-68.
19. Vaquero MJR, Serravalle LRT, de Nadra MCM and de Saad AMS: Antioxidant capacity and antibacterial activity of phenolic compounds from Argentinean herbs infusions. Food Control 2010; 21: 779-85.
20. Narsih, Kumalaingsih SS, Usinggih W and Wignyanto: Microencapsulation of natural antioxidant powder from Aloe vera (L.) skin using foam mat drying method. International Food Research Journal 2013; 20: 681-85.
21. Datta A, Ghoshdastidar S and Singh M: Antimicrobial property of piper betel leaf against clinical isolates of bacteria. International Journal of Pharmaceutical Sciences and Research 2011; 2: 104-09.
22. Wang Y, Lu ZX, Wu H and Lv FX: Study on the antibiotic activity of microcapsule curcumin against foodborne pathogens. International Journal of Food Microbiology 2009; 136: 71-74.
23. Pinelo M, Arnous A and Meyer AS: Upgrading of grape skins: Significance of plant cell-wall structural components and extraction techniques for phenol release. Trends in Food Science & Technology 2006; 17: 579-90.
24. Yang YC, Li J, Zu YG, Fu YJ, Luo M, Wu N and Liu XL: Optimisation of microwave-assisted enzymatic extraction of corilagin and geraniin from Geranium sibiricum Linne and evaluation of antioxidant activity. Food Chemistry 2010; 122: 373-80.
25. Criado MR, Torre SP, Pereiro IR and Torrijos RC: Optimization of a microwave-assisted derivatization-extraction procedure for the determination of chloro-phenols in ash samples. Journal of Chromatography A 2004; 1024: 155-63.
26. Zhou HY and Liu CZ: Microwave-assisted extraction of solanesol from tobacco leaves. Journal of Chromatography A 2006; 1129: 135-39.
27. Lay-Keow N and Michel H: Effects of moisture content in cigar tobacco on nicotine extraction similarity between Soxhlet and focused open-vessel microwave-assisted techniques. J of Chromatography A 2003; 1011: 213-19.
28. Proestos C and Komaitis M: Application of microwave-assisted extraction to the fast extraction of plant phenolic compounds. LWT-Food Science and Technology 2008; 41: 652-59.
29. Hemwimon S, Pavasant P and Shotipruk A: Microwave-assisted extraction of antioxidative anthraquinones from roots of Morinda citrifolia. Separation and Purification Technology 2007; 54: 44-50.
30. Zhang HF, Yang XH, Zhao LD and Wang Y: Ultrasonic-assisted extraction of epimedin C from fresh leaves of Epimedium and extraction mechanism. Innovatvie Food Science and Emerging Technologies 2009; 10: 54-60.
31. Zhang QA, Zhang ZQ, Yue XF, Fan XH, Li T and Chen SF: Response surface optimization of ultrasound-assisted oil extraction from autoclaved almond powder. Food Chemistry 2009; 116: 513-18.
32. Mukataka S, Tada M and Takahashi J: Effects of agitation on enzymatic hydrolysis of cellulose in a stirred-tank reactor. J of Fermentation Technology 1983; 61: 615-21.
33. Ingesson H, Zacchi G, Yang B, Esteghlalian AR and Saddler JN: The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose. Journal of Biotechnology 2011; 88: 177-82.
34. Wright JD: Ethanol from biomass by enzymatic hydrolysis. Chem Eng Progress (United States) 1988; 84.
35. Gan Q, Allen SJ and Taylor G: Kinetic dynamics in heterogeneous enzymatic hydrolysis of cellulose: an overview, an experimental study and mathematical modelling. Process Biochemistry 2003; 38: 1003-18.
36. Dangles O: The physico-chemical properties of polyphenols-How do they relate to their roles in plants, foods and human health. Agro Food Industry Hi-tech 2006; 17: 64-67.
37. Moosavy MH, Hassanzadeh P, Mohammadzadeh E, Mahmoudi R, Khatibi SA and Mardani K: Antioxidant and antimicrobial activities of essential oil of Lemon (Citrus limon) peel in-vitro and in a food model. Journal of food quality and hazards control 2017; 4: 42-48.
38. Pan YM, He CH, Wang HS, Ji XW, Wang K and Liu PZ: Antioxidant activity of microwave-assisted extract of Buddleia officinalis and its major active component. Food Chemistry 2010; 121: 497-502.
39. Munin A and Edwards-Lévy F: Encapsulation of natural polyphenolic compounds; a review. Pharmaceutics 2011; 3: 793-829.
40. Desai KGH and Jin Park H: Recent developments in microencapsulation of food ingredients. Drying Technology 2005; 23: 1361-94.
41. Anjaneyulu E, Ramgopal M, Hemalatha S and Balaji M: Phytochemical analysis, anti microbial and anti oxidant activity of the bark extracts of Acacia leucophloea Global Journal of Biotechnology and Biochemistry 2010; 5: 231-36.
42. Afzal J, Ullah N, Hussain Z, Rukh S, Ayaz M, Akbar A and Zaman A: Phytochemical analysis and antibacterial potential of leaf extract of Bauhinia: an ethnomedicinal plant. Matrix Science Pharma 2017; 1: 17-19.
    

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    How to cite this article:

    Ayyasamy S and Velusamy S: Enhancing the extraction of polyphenols from Acacia leucophloea bark by microwave-assisted enzymatic extraction method and in-vitro study of its antioxidant and antibacterial activity. Int J Pharm Sci & Res 2021; 12(6): 3320-30. doi: 10.13040/IJPSR.0975-8232.12(6).3320-30.

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