Phenolic compounds from Syzygium cumini (L.) Skeels leaves: Extraction and membrane purification

Journal of Applied Research on Medicinal and Aromatic Plants 12 (2019) 43–58

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Phenolic compounds from Syzygium cumini (L.) Skeels leaves: Extraction and membrane purification Upasna Balyan, Satya Pal Verma, Biswajit Sarkar

T

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University School of Chemical Technology, GGS Indraprastha University, Delhi, 110078, India

ARTICLE INFO

ABSTRACT

Keywords: Syzygium cumini(L.) Skeels leaf Polyphenol Response surface methodology Microfiltration Storage stability

A hybrid process consisting of extraction and microfiltration was proposed in this study for producing purified, clear and stable aqueous phenolic extract from jamun (Syzygium cumini (L.) Skeels) leaf. Response surface methodology was successfully used for optimization of extraction conditions. Pseudo-first order kinetic model successfully described the extraction of total polyphenols from jamun leaves, with the activation energy determined as 9.5 kJ/mol based on the Arrhenius model. The kinetic constants were used to study the kinetic and thermodynamic compensations of extraction of TPC from jamun leaves. Applying the statistical criterion, the kinetic and thermodynamic compensations were concluded to be real and the extraction process was controlled by entropy. A total of ten phenolic compounds including six phenolic acids (tannic acid, gallic acid, ellagic acid, caffeic acid, ferulic acid and p-coumaric acid) and four flavonoids (catechin, epicatechin, quercetin and myricetin 3-O-rhamnoside) were identified and quantified in jamun leaf extract obtained under optimum extraction conditions. The selection of appropriate membrane in the microfiltration step was a critical aspect. To observe the effect of membrane pore size on the permeate flux and permeate quality, leaf extracts were then microfiltered using four different microfiltration membranes (0.1, 0.22, 0.45, and 0.8 μm) under batch concentration mode. The flux decline was successfully described by the Hermia’s cake filtration model. The stability of clarified extract was investigated at 4 °C for 45 days. The 0.45 μm microfiltration membrane was suggested for the clarification of jamun leaf extract in order to achieve high flux, polyphenol recovery, extract purity and improved storage stability.

1. Introduction Syzygium cumini (L.) Skeels (Jamun), Myrtaceae family plant, is best known for high nutritional value. Agricultural by-products of jamun plant, particularly leaves are traditionally used in ayurvedic medicine to treat diabetes, gallbladder stones and for strengthening teeth and gums etc. Many of these potential health benefits are attributed to polyphenolic compounds having antioxidant properties (Swami et al., 2012). For extraction of plant polyphenols, a lot of research has been focused on agricultural waste, particularly leaves of many plants such as, passiflora alata Curtis, borage, pitanga, smilax, curry (Colomeu et al., 2014; Martinez-Correa et al., 2011; Ningappa et al., 2008; Ozsoy et al., 2008; Segovia et al., 2015) etc. In fact, the discarded jamun leaves can be cheap and abundant raw material to produce natural antioxidants because of plentiful bioactive compounds in them (Kaneria and Chanda, 2013). Therefore, it is important to investigate and exploit the jamun leaves as a resource of natural antioxidants. Profiling of phenolic compounds in any plant leaves is generally carried after

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extraction of phenolic compound using suitable solvent. The extract quality and extraction efficiency are normally affected by several factors such as the extraction technique, type of solvent used and its concentration, the liquid-solid ratio, time and temperature of extraction, and particle size of the solid matrix etc. (Dahmoune et al., 2015). In the present study, conventional solid–liquid extraction of dried jamun leaf powder is performed using water as solvent, due to its wide use in extraction of phenolic compounds from various plant leaves (Sousa et al., 2016; Kumar et al., 2012). No detailed information is available in the literature about the optimal extraction conditions to obtain the aqueous extract of jamun leaves with maximum extraction yield, extract purity and antioxidant activity, simultaneously. This has led us to study on the optimization of process parameters for aqueous extraction of phenolic compounds from jamun leaves. Furthermore, several equations have been suggested in the literature to model solidliquid extraction kinetics of phenolic compounds from plant material (Khan et al., 2010; Minchev and Minkov, 1984; PELEG, 1988; Qu et al., 2010). However, no such studies about the aqueous extraction kinetics

Corresponding author. E-mail addresses: [email protected], [email protected] (B. Sarkar).

https://doi.org/10.1016/j.jarmap.2018.12.002 Received 28 February 2018; Received in revised form 6 November 2018; Accepted 26 December 2018 Available online 15 January 2019 2214-7861/ © 2019 Elsevier GmbH. All rights reserved.

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Nomenclature At A0 Ct C∝ Ea K k kb kc kI ks kisokin keq kisoeq k1 k2 k0

n R R2 T t V0 VP VR Vw V 0w V fw X Y ΔS# ΔH# ΔG# ΔS ΔH ΔG

Concentration of TPC/TFC at storage time t, mg/L Initial cconcentration of TPC/TFC during storage, mg/L Concentration of TPC at operation time t, mg GAE/ 100 g leaf Concentration of TPC at saturation, mg GAE/ 100 g leaf Activation energy (J mol−1) Degradation rate constant during storage (week −1) Deterioration rate constant (s−1) Complete blocking constant (s−1) Cake filtration constant (s m−2) Intermediate blocking constant (m−1) Standard blocking constant (m−0.5s−0.5) Isokinetic rate constant, min−1 Equilibrium rate constant, min−1 Isoequilibrium rate constant, min−1 Peleg’s rate constant, 100 g leaf. min/mg GAE Peleg’s capacity constant, 100 g leaf/mg GAE Frequency factor (s−1)

Blocking index, dimensionless Universal gas constant, (J mol−1 K−1) Coefficient of correlation, dimensionless Temperature, (0C/K) Filtration Time, sec. Solute volume in feed, mL Solute volume in permeate, mL Solute volume in retentate, mL Permeate flux, L/m2 h Initial permeate flux, L/m2 h Final permeate flux, L/m2 h Real variables Response variable Entropy of activation (J mol−1 K−1) Enthalpy of activation (J mol−1) Free energy of activation (J mol−1) Changes in entropy (J mol−1 K−1) Changes in enthalpy (J mol−1) Changes in free energy (J mol−1)

extract with different pore size of microfiltration membrane. In addition the stability of original leaf extract was compared with MF-clarified leaf extract during storage at 4 °C for 45 days and finally an appropriate MF membrane was selected for clarification of jamun leaf extracts.

of jamun leaves have been reported. The kinetic study seems to be incomplete without the determination of extraction process. The study of kinetic and thermodynamic compensations can provide better description of the extraction and its process whether it is controlled by enthalpy or entropy. The crude aqueous phenolic extract contains some polysaccharides (cellulose, pectins etc.) which are co-extracted along with phenolic compounds leading to decrease in purity of the extract. For this reason, the present work is also focused in membrane processing as green technology for purification of jamun leaf extract. Ultrafiltration (UF) and microfiltration (MF) have been successfully employed for purification of phenolic compounds extracted from various plant leaves (Sousa et al., 2016; Todisco et al., 2002; Torun et al., 2014). MF membranes retain large molecular species like polysaccharides to enhance the permeate quality and clarity. Nevertheless, the major problem which hinders more widespread applications of MF is the decline in permeate flux with time due to membrane fouling. These macromolecules are responsible for development of concentration polarization near the membrane surface, pore blocking and cake/gel type layer formation on the membrane surface leading to shortening of membrane life and increase in operational cost (Cheryan, 1998; Porter, 2005). Therefore, understanding of appropriate flux decline mechanism is important for efficient design and scale up the filtration systems. Among the several models attempted, Hermia’s pore blocking model has been successfully used to explain the flux decline of tea leaf extract (Sousa et al., 2016). Moreover, to our knowledge, there is no investigation available to test the Hermia’s models for identification of fouling mechanisms and quantification of transient flux decline during ultrafiltration of jamun leaves extract. With regard to stabilization of extract, it would be interesting to note that clarification enhances the shelf life of the extract by removing the high molecular impurities such as cellulose, polysaccharides etc. which are responsible for microbial spoilage or degradation of antioxidant constituents during storage (Mondal et al., 2016). The objectives of this study are: (a) to optimize the extraction condition using RSM, in order to maximize the extraction yield, extract purity and antioxidant activity, simultaneously, (b) to identify and quantify the individual phenolic compounds in the jamun leaf extracts, (c) to study the extraction kinetics of polyphenols and to test the applicability of literature reported extraction models, (d) to study the kinetic and thermodynamic compensations for identification of extraction mechanism (entropic or enthalpic), (e) to identify the flux decline mechanism by Hermia’s model during clarification of phenolic

2. Methodology 2.1. Materials Dried jamun leaf powder was provided by M/s. Dev Bharti Ausdhalaya Kendra, Uttarakhand, India. According to sources, jamun leaves were loaded on stainless trays and dried in a circulating air oven at 50 °C until it attained constant weight. The dried leaves were crushed and passed through 100-mesh sieve. The dried powder leaves were packed in sealed plastic bags and shipped to the Separation laboratory (USCT, GGSIP University, Delhi), where they were stored at 4 °C. The dried leaf powder that was sieved through a 140-mesh (0.105 mm) sieve was used for polyphenol extraction studies. Since the varying moisture content in leaf could affect the extraction yield of polyphenols, the dried leaf powder was used for extraction of polyphenols. Distilled water was used as the solvent for the extraction process. The chemicals, 1,1-diphenyl-2-picrylydrazyl (DPPH), 2, 4, 6- tripyridyl-s-triazine (TPTZ), Folin–Ciocalteu phenol reagent, (+)-catechin hydrate (≥95%), (-)-epicatechin (≥90%), caffeic acid (≥98%), p-coumaric acid (≥98%), ellagic acid (≥95%), quercetin (≥95%), myricetin 3-Orhamnoside (≥99%), ferulic acid (≥95%), Triethanolamine, and tannic were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO). Bovine serum albumin, Gallic acid, ascorbic acid, glacial acetic acid, sodium acetate trihydrate, butylated hydroxytoluene (BHT), potassium acetate, methanol, ferrous sulphate, sodium hydroxide, aluminium chloride and potassium acetate were obtained from SRL (SRL Pvt. Ltd., India). All other chemicals used were of analytical grade. Four flat sheet MF membranes with an average pore size of 0.1 μm (cellulose acetate), 0.22 μm (polyvinyldene fluoride), 0.45 μm (polyvinyldene fluoride), and 0.8 μm (cellulose acetate) obtained from Millipore (India) Pvt. Ltd. were used in this study. The hydraulic permeability of 0.1, 0.22, 0.45 and 0.8 μm membranes were experimentally found to be (4.4 ± 0.2)×10−10, (6.7 ± 0.5)×10-9, (19.5 ± 0.6)×10-9 and (28.5 ± 0.8)×10-9 m/Pa-s, respectively. 2.2. Experimental design Response surface methodology (RSM) with full factorial central 44

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composite design at five levels of each variable was employed to determine the optimal extraction conditions (Khuri and Cornell, 1987). The selected independent variables were extraction temperature (X1, 34.8–85.2 °C), extraction time (X2, 49.7–100.2 min), L/S ratio (X2, 9.77–60.2 mL/g). The extract purity (Y1), extract yield (Y2), and the antioxidant capacity measured by DPPH radical scavenging activity (Y3), were selected as responses. A second-order polynomial model was used to predict the responses as (Montgomery, 2009), 4

Y=

o

+

4 i Xi

i= 1

+ i= 1

2 ii X i

3

change in agitation. On the contrary, when a reaction is controlled by entropy, the changes in the frequency factor are higher than the changes in the activation energy. Thus, the extraction process is less sensitive to changes in the temperature and more sensitive to such entropic changes as agitation. For solid-liquid extraction process, Arrhenius model was commonly used to describe extraction rate to temperature relations followed by estimation of activation energy (Ea). Temperature dependence of the extraction kinetics could be expressed by Arrhenius equation as (Ibarz et al., 2017),

4

+

ij Xi Xj i= 1 j= i+1

+

(1)

1nk = 1n(k 0)

Where, Y was the response variable and the coefficient of the polynomial were represented by β0 (constant or offset term); βi (linear effects); βii (quadratic effects); βij (interaction effects). Xi were independent variables and was the error. The experimental design was carried out using ‘Design Expert’ software (Version 8.0.6, Stat-Ease, Inc., Minneapolis, USA). The experimental design consisted of 20 runs including six replications at the center point (60 °C, 75 min, 35 mL/g).

Ea RT

(2) −1

−1

Where, k0 was a constant (mg GAE g min ), R the universal gas constant (8.314 J mol−1 K−1), Ea the activation energy (J mol−1) and T was the absolute temperature (K). The values of frequency factor and the activation energy were obtained from the Arrhenius equation (Eq. 2) for different values of an environmental variable (liquid-to-solid ratio in this case). The kinetic compensation consisted of the linear relationship between the logarithm of the frequency factors (lnk0) and the activation energies (Ea) as (Ibarz et al., 2017),

2.3. Extraction

ln(k o) = C + D(Ea)

Extraction process was performed under constant stirring of 700 rpm using Teflon-coated magnetic stirrer (Remi 5 MLH) in a flat bottom flask (1 L capacity) connected to a condenser. Dried jamun leaf powder (10 g) was placed in the flat bottom flask with pre-estimated volume of distilled water at the desired temperature according to experimental design. The initial time was set when jamun leaf powder was added to the water. At the end of the extraction, the extract was allowed to cool at room temperature (25 ± 1 °C) and filtered through Whatman No. 1 paper to separate the residual leaf material and liquid extract was stored at 4 °C for subsequent analysis. Furthermore, extraction kinetics of total polyphenols content were evaluated by withdrawing extract samples of 5 mL at various predetermined times (2, 4, 6, 8, 10, 15, 30, 50, 60, 70, 90 min) replacing the extract volume with distilled water up to 90 min for L/S ratio of 35:1 at the temperatures of 34.8 °C, 60 °C and 85 °C and at temperature of 60 °C for L/S ratios of 9.8:1, 35:1 and 60.2:1. The solids residue left after filtration was dried at 40 ± 1 °C until constant weight in hot air oven for preparing samples for SEM analysis. Then the filtrate was used for further clarification purpose.

(3)

Where, C and D were the constants. Eq. (3) implied that any change in activation energy was accompanied by the proportional change in logarithm of the frequency factors. As both parameters contributed with opposite sign in Eq. (2), the effect on the rate constant of the change in Ea was partially compensated by the effect of the change in the lnk0 (Garvín et al., 2017). Kinetic compensation indicated the existence of the isokinetic temperature (Tisokin), this being the temperature at which all the rate constants had the similar value. Comparing Eq. (2) and (3), the Tisokin and rate constant (kiso) at Tisokin could be obtained as,

Tisokin =

1 DR

kisokin = Exp (C)

(4) (5)

Applying transition state theory in extraction process, the kinetic constant (k) could be related to the equilibrium constant (keq) as,

k=

2.4. Kinetics and thermodynamic compensation

kBT k eq h

(6)

Where, kB was the Boltzmann’s constant (1.38 × 10−23 J K−1) and h was the Plank’s constant (6.626 × 10−34 J s). Furthermore, according to the Van't Hoff equation, the equilibrium constant could be written to the variation of activation enthalpy (ΔH#) and activation entropy (ΔS#) as (Garvín et al., 2017),

The study of kinetic and thermodynamic compensations can provide better description of the extraction process whether it is controlled by enthalpy or entropy. The compensation effects have been studied in many physical, chemical, biological and food processes and have been reviewed in details (Garvín et al., 2017). In the present study, all the kinetic data obtained from most appropriate kinetic model (pseudo-first order model in this case), are evaluated using kinetic and thermodynamic compensations, to identify the control extraction process (entropic or enthalpic) occurring during extraction of TPC from jamun leaves. Any change in the environmental variable (liquid-to-solid ratio in this case) causes changes in both the frequency factor and the activation energy (Ibarz et al., 2017). The frequency factor is an entropic factor signifying the rate of collisions among the molecules and therefore, depends on the agitation level of the extraction media. However, the activation energy is related to the activation enthalpy, representing the minimal energy that a specific collision between the molecules must achieve in order to allow the extraction process to take place. The activation energy can also be understood as the sensitivity of the kinetic constant to the temperature. When an extraction process is controlled by enthalpy, the changes in the activation energy are proportionally greater than the changes in the frequency factor. It means that the extraction is more sensitive to the change in the temperature than the

ln(k eq) =

S# H# R RT

(7)

Thus, a plot of the logarithm of the equilibrium constant against the reciprocal absolute temperature was linear. The values of ΔH# and ΔS# were estimated from the slope (-ΔH#/R) and intercept (ΔS#/R), respectively, for different values of L/S ratio. Thermodynamic compensation took place when the activation enthalpy variation (ΔH#) could be related with the activation entropy variation (ΔS#) through a linear relationship as,

H# = p+ q( S#)

(8)

Thermodynamic compensation could be explained by the fact that a stronger intermolecular interaction or bonding (associated with the enthalpy) led to decrease in configurational freedom resulting in greater order of the system (associated with the entropy). The changes in activation Gibbs free energy (ΔG#) could be obtained from equilibrium rate constant (Keq) as, 45

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G# = -RT1nk eq

(9)

Extraction yield(%) =

Thermodynamic compensation implied the existence of the isoequilibrium temperature (Tisoeq), this being the temperature at which all the equilibrium constants values were similar. Comparing Eq. (7) and (8), the Tisoeq and kisoeq could be obtained as, Tisoeq = q

ln(kisoeq) =

(13)

Purity(%) =

(10)

-p RTisoeq

(11)

n n 1 i= 1 Ti

Concentration of TPC in the extract x100 Concentration of TS in the extract

(14)

For analysis, all measurements were done in triplicate and results were presented as mean ± standard deviation. Determination of antioxidant activities (AA) The antioxidant activity of jamun leaf extract was assessed by DPPH scavenging assay according to the method of McCune and Johns, 2002 with minor modifications, which was based on the reaction of antioxidant compound with DPPH free radical in methanol solution. The reduction capacity of the extract (on the DPPH radical) was determined by the decrease in its absorbance at 715 nm. The antioxidant activity was calculated as percentage inhibition. The antioxidant activity was inversely proportional to IC50 value (50% inhibitory concentration), which was calculated from the linear regression of the % inhibition curves obtained for varying concentrations of dried extract. The AA of leaf extract was also determined by the Ferric-reducing ability power (FRAP) spectrophotometric method, following the procedure described by Gohari et al. (2011) with some modifications. The FRAP assay was based on the ability of the antioxidants in the extract to reduce ferrictripiridyl-triazine complex [Fe+3-TPTZ] to its ferrous form [Fe+2TPTZ] in an acidic condition. The results were compared with a calibration curve of standard ferrous sulphate (FeSO4.7H2O; 50–500 μmol/ L) solution and expressed in μmol Fe(II)/L. All measurements were performed in triplicate samples.

To confirm the existence of compensation effect, experimental harmonic mean temperature (Thm) was generally calculated and compared to Tiso. According to test of Krug (Krug et al., 1976), a linear compensation effect existed only if the Tiso was different from the Thm, which was defined as,

Thm =

Weight of TS in the extract x100 Weight of leaf powder taken for extration

(12)

If Thm was outside the confidence interval of Tiso, a real compensation could be concluded with confirmed effect of the environmental variable (L/S ratio). However, if Thm was within the confidence interval of Tiso, the linear relationship observed in (lnk0 - Ea) or (ΔH# - ΔS#) was the consequence of the propagation of experimental errors and hence, the observed compensation was actually statistical compensation. The control extraction process changed from enthalpic to entropic at Tiso. According to Leffler’s criterion (Leffler, 1955), if Tiso > Thm, the process was enthalpically controlled, and, on the other hand, if Tiso < Thm, the process was controlled by entropy. 2.5. Analysis of leaf powder by SEM

2.7. High-performance liquid chromatography (HPLC) analysis

To observe the effect of extraction treatment on the surface morphology of leaf particles scanning electron microscopy (SEM) (EVO 18 Research, ZEISS, Germany) was used after coating samples with platinum using a physical vapor deposition method with a sputter coater (AGAR Sputter Coater, UK) and the respective micrographs were shown in Fig. 2. The SEM images were processed using Image J software to obtain the particle sizes. The mean particle size and particle size distribution histogram of leaf particles were obtained using OriginPro 8.0 software.

Perkin Elmer 200 series (USA) HPLC equipped with an autosampler ABI 785 detector was employed to identify and quantify the individual phenolic acids and flavonoids present in the leaf extract. For quantitative determination of seven phenolic compounds (gallic acid, ellagic acid, caffeic acid, p-coumeric acid, catechin, epicatechin, and quercetin), chromatographic separation was performed at a wave length of 280 nm using C18 (Atlantis T3) column at 25 ± 1 °C, based on the method described by Balyan and Sarkar (2017). The quantitative determination of myricetin 3-O-rhamnoside and ferulic acid in the leaf extract was carried out using the same column at 25 ± 1 °C under isocratic mode at wavelength of 256 nm and 321 nm, respectively. For ferulic acid, extract samples were eluted with the mobile phase consisting a mixture of 78:22 (v/v) acetonitrile (solvent A) and a mixture of acetonitrile/ glacial acetic acid (15:5, v/v) (solvent B). For myricetin 3O-rhamnoside, extract samples were eluted with the mobile phase consisting a mixture of 78:22 (v/v) 0.1% H3PO4 (solvent A) and acetonitrile (solvent B). Phenolic compounds in the leaf extract were identified by comparing the retention time and their spectral characteristics against those of standards under same conditions. Quantitation of phenolic compounds was made according to the linear calibration curves of the corresponding standard compounds.

2.6. Analytical assay Total phenolic content (TPC) in the aqueous jamun leaf extract was determined using modified Folin–Ciocalteau colorimetric method described previously (Singleton and Rossi, 1965; Balyan and Sarkar, 2017). Results were expressed as mg of gallic acid equivalent (GAE) per gram of dried extract. The total flavonoid content (TFC) in the extract was determined using aluminium chloride colorimetric method (Balyan and Sarkar, 2017; Chang et al., 2002) using quercetin as standard and with absorbance measurements at 415 nm using a UV spectrophotometer. The results were expressed in mg quercetin per gram of dried extract. Tannic acid in the extract was determined by the protein precipitation method described previously (Balyan et al., 2016; Hagerman and Butler, 1978) and expressed in μg tannic acid/ mL of extract. Color and clarity of the extract was monitored by absorbance at 420 nm and transmittance at 660 nm, respectively using UV/VIS Spectrophotometer (U-2900, Hitachi, Japan). Alcohol-insoluble-solids (AIS) present in the extract was determined by alcohol precipitation technique as described by Hart and Fisher (2012) and expressed in percentage by weight. The total solids (TS) content of the extract sample was measured gravimetrically by drying the extract in a hot air oven at 85 ± 1 °C (Ranganna, 1986). The extraction yield and purity of the extract were determined as,

2.8. Statistical analysis Analysis of variance (ANOVA) was performed to estimate the statistical significance of each independent variable and their interactions to the prediction of dependent variables. The response surface plots were drawn in order to visualize the effect of independent variables on dependent variable. The optimal extraction conditions according to model were determined by simultaneously maximizing extract yield, purity and antioxidant activity with the help of Design Expert’ software. 46

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Fig. 1. Response surface for the effects of: temperature and L/S ratio (a), and time and L/S ratio (b), on the extract purity; temperature and L/S ratio (c), and time and L/S ratio (d), on the extraction yield; temperature and L/S ratio (e), and time and L/S ratio (f), on DPPH.

47

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Fig. 2. SEM images of jamun leaf powder: (a) before extraction (×1000), (b) after extraction (×1000), (c) before extraction (×5000), (d) after extraction (×5000); Histogram particle size distribution: (e) before extraction, (f) after extraction.

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2.9. Clarification of the leaf extracts

indicating that treatments were significantly different. The ANOVA results demonstrated that the values of coefficient of determination (R2), adjusted determination coefficient (Adj. R2) and coefficient of variation (CV) were found to be 0.956, 0.916 and 3.05%, respectively for Y1; 0.911, 0.83 and 4.83%, respectively for Y2; and 0.984, 0.969 and 8.16%, respectively for Y3. Higher values of R2 and Adj. R2 exhibited a close agreement between the experimental and the predicted values while lower value of C.V. clearly indicated the reliability of the experimental values with high degree of precision. The three-dimensional response surface plots based on the experimental data were demonstrated in Fig.1(a–f) in order to study the interactive effect of the process variables on the response. The probability level of regression analysis indicated that the linear terms of L/S ratio (p < 0.001), extraction temperature (p < 0.01) and extraction time (p < 0.05) were significant with respect to extract purity. The interaction term between extraction time and L/S ratio as well as between extraction temperature and L/S ratio were found significant at the probability level of p < 0.01 whereas, the quadratic terms of extraction temperature were also found significant (p < 0.01). As can be seen in Fig. 1(a) that the extract purity increased significantly with the increase of extraction temperature from 34.8 to 50 °C and L/S ratio from 9.8:1 to 50:1 mL/g while keeping extraction time constant. On further increase in L/S ratio, no significant change in purity was observed. However, temperature beyond 50 °C resulted in lower extract purity. Fig.1b showed that extract purity increased with increasing time up to 85 min. Thereafter, there was a decrease in extract purity. Moreover, higher extraction temperature with longer extraction time led to decrease in TPC of the extract. Therefore, extract purity declined. Temperature and L/S ratio were also found as the key parameters which influenced the extraction yield significantly. Increasing temperature improved the extraction yield which might be due to enhanced solubility and diffusion coefficient of antioxidants at a high temperature (Amendola et al., 2010). Extraction yield increased with an increase in L/S ratio. It was obvious from the mass transfer principle, an increase in liquid volume in the system resulted to an increase in diffusion due to increase in concentration gradient between the solid and liquid (Cacace and Mazza, 2003). The effects of L/S ratio and extraction temperature shown in Fig.1e illustrated that the antioxidant activity of the extract increased with increase in L/S ratio as well as with temperature up to a certain value as observed in case of extract purity. Higher extraction temperature showed a negative effect on the antioxidant activity. This might be explained by the fact that phenolic compounds are thermosensitive substances which underwent degradation at higher temperature and leading to decrease in antioxidant activities. Maximum antioxidant activity was obtained at L/S ratio of 50 and temperature of 85 °C when the time was fixed at 80 min. This observation was further confirmed by the results of ANOVA which showed that the linear effects of extraction temperature (p < 0.001) and L/S ratio (p < 0.0001) along with quadratic term of extraction temperature (p < 0.05) were significant for the response of antioxidant activity. The relationship between dependent variables and independent variables were represented in the following equations:

Aqueous jamun leaf extracts obtained under optimal extraction condition, with initial feed volume of 400 mL, was subjected to clarification using microfiltration (MF) at room temperature of 25 ± 1 °C. During clarification, four flat sheet MF membranes with an average pore size of 0.1 μm, 0.22 μm, 0.45 μm, and 0.8 μm were used. The effective filtration area was 32.15 cm2. MF experiments were conducted in a stirred cell (inner diameter: 76 mm, capacity: 500 mL), under constant pressure and stirrer speed of 138 kPa and 1000 rpm, respectively, for the duration of 60 min. Nitrogen gas was used to generate pressure in the stirred cell. The detailed description of the experimental set up was shown elsewhere (Sarkar, 2013). In the actual experiment, permeate was collected from the bottom of the cell in a measuring cylinder. Volume reduction ratio was calculated as, VRR(%) = (Vp/Vo)x100 ; where, V0 and VP were the feed volume and permeate volume, respectively. VRR indicated the percent of initial feed volume permeated (Cheryan, 1998). The clarified leaf extracts were then used for subsequent analysis. Prior to clarification, all membranes were compacted at 207 kPa. 2.10. Identification of fouling mechanism To obtain an economically feasible clarification process, it was very important to understand and analyze the membrane fouling during ultrafiltration of jamun leaves extract. Hermia’s pore blocking models were used to identify the fouling mechanism (Hermia, 1982). Hermia developed an empirical model for constant pressure dead-end filtration of non-Newtonian fluid as,

d2t dt =K dV 2 dV

n

(15)

Where, V and t were the cumulative volume of filtrate and filtration time, respectively. The blocking index, n was a dimensionless number that characterizes the fouling mechanism. This model could be extended to Newtonian fluids when the power law index was substituted by 1. Eq.(15) could be expressed in terms of permeate flux as,

dVw = - kV3-n w dt

(16) 1 dV . A dt

Where, permeate flux, Vw = The resistance coefficient, k was dimensional and depends on the system, the membrane, and the operating conditions. The value of ‘n’ in Eq. (16) was fixed at 0, 1, 1.5 or 2 to represent the fouling mechanisms such as cake filtration, intermediate blocking, standard blocking, and complete pore blocking, respectively. Eq.(16) could be linearized for different values of ‘n’ (Lim and Bai, 2003) and were used to identify the fouling mechanism and further to explain flux decline behavior with time in the microfiltration of jamun leaves extract under batch concentration mode. 2.11. Storage study Liquid samples of original jamun leaf extract and MF-clarified extracts obtained with the membranes of 0.1, 0.22, 0.45 and 0.8 μm were stored in closed amber sterilized glass bottles in the dark at refrigerated temperature (4ºC) for 45 days. In order to evaluate the extract stability, samples were analyzed every 7 days with respect to TPC, TFC, color, and clarity. All measurements were done in triplicate. Results were reported as mean ± standard deviation of two independent determinations.

Y1 = 27.22 + 0.66X1- 0.6X2 + 2.22X3- 1.3X1X2 + 1.738X1X3+ 0.62X2X3- 0.39X12 - 0.52X22- 0.98X32

(17)

Y2 = 20.19 + 0.21X1- 0.23X2+ 2.08X3- 0.57X1X2- 1.05X1X3- 0.037X2X3- 0.38X12 + 0.48X22- 0.97X32

(18)

Y3 = 82.37 - 4.81X1- 6.04X2- 0.33X3+ 0.68X1X2+ 17.28X1X3- 1.41X2X3 - 16.49X12- 3.52X22- 20.42X32

3. Results and discussion

(19)

After numerical optimization, L/S ratio of 42.7:1 (v/w), extraction temperature of 68.3 °C and time of 61 min were found to be optimal conditions. Under these optimal conditions, the model predicted responses were: purity (29.6%), extraction yield (21.3%), and DPPH

3.1. Statistical analysis The responses were highly significant at the 99% confidence level, 49

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radical scavenging activity (76.8%). In order to verify the predictive capacity of the response surface methodology model, experiments were performed under the optimal conditions in triplicate. Aqueous jamun leaf extract obtained under the optimal extraction conditions was analyzed for TPC, TFC, AIS, purity, color, clarity, pH, viscosity, DPPH, FRAP, and IC50 etc. The measured values were: purity (28.0 ± 0.5)%, extraction yield (20.9 ± 0.5)%, and DPPH radical scavenging activity (82.5 ± 1.5)%. The experimental results were in good agreement with predicted values demonstrating the validation of the model with a good correlation for the extraction of polyphenols from jamun leaves. The obtained results were better than those reported by Kaneria and Chanda, (2013) in the extraction of phenolic compounds from jamun leaves using different solvents, achieving TPC of 21.77% and 10.44% of extracted compounds using solvent as acetone and water, respectively. This difference could be related with particle size of the leaves, extraction conditions, harvesting time and the location of the plant. The TPC of the aqueous jamun leaves extract (1370 ± 41.7 μg/mL or 58.5 ± 1.8 mg GAE/g leaves) was comparable with the values reported for other aqueous plant leaves extract such as curry (54 ± 4.4 mg GAE/g leaves) (Ningappa et al., 2008), smilax (30.6 mg GAE/g leaves) (Ozsoy et al., 2008), pitanga (28 mg GAE/g leaves) (Martinez-Correa et al., 2011), passiflora alata curtis (9.5 ± 2.8 mg GAE/g leaves) (Colomeu et al., 2014), green tea (1.73 mg GAE/g leaves) (Sousa et al., 2016), and borage (0.09 mg GAE/ g leaves) (Segovia et al., 2015), at different time and temperature combinations for extraction. The DPPH radical scavenging activity of jamun leaf extract was higher than the aqueous extract of curry leaves (41% inhibition), stevia leaves (52.46% inhibition) at the concentration of 100 μg/mL (Ningappa et al., 2008; Rao Narsin et al., 2014). It was observed that aqueous extract was acidic in nature (pH 5.23). The AIS content of the extract was found to be 2520 ± 71 μg/mL which might be responsible for lower clarity (49.5%) of the extract. Total flavonoid content (133 μg QE /mL) accounted for 9.7% of the total amount of polyphenol extracted. Jamun leaf extract also exhibited both DPPH

radical scavenging abilities as well as ferric reducing antioxidant power. The IC50 value of the jamun leaf extract (the concentration of phenolic compounds required to quench 50% of the initial DPPH) was estimated as (54.5 ± 1.65) μg/mL. Furthermore, the leaf extract also presented antioxidant activity of 338 ± 10 μM Fe(II)/L by FRAP method. 3.2. Effect of extraction treatment on morphology of leaf powder Figs.2(a–d) showed images obtained by scanning electronic microscopy (SEM) of the sample of jamun leaf powder before and after aqueous extraction with a magnification factor of 1000x and 5000 × . The samples were observed using an accelerating voltage of 20 kV. SEM study showed that leaf powder consisted of non-uniform, irregularshaped particles with different sizes. Figs.2(e–f) showed the size distribution histogram of leaf particles before and after extraction, respectively. As observed in Fig.2e, the size of particles was found to be 0.3–80 μm with the maximum number of particles (77%) in the size range of 0.3–5 μm. The average diameter of leaf particle was found to be 4.42 ± 0.025 μm. After extraction, leaf tissue became porous with hollow openings resulting in increase in average particle size. During extraction, an increasing number of leaf structures were uncovered. The average diameter of leaf particle after extraction, was found to be 8.37 ± 0.046 μm, in the size range of 0.3–100 μm (Fig.2f). The appearance of openings in the leaf tissues proved the efficiency of aqueous extraction which destructed the basic shapes. The change in morphology of the leaf due to extraction was more evident at high magnification (5000x). These results were in agreement with those found by Dahmoune et al., (2015). Aqueous extraction was based on the diffusion of the water into the solid matrix and extraction of the phenolic compounds within the cells into the surrounding water by solubilization (Ballard et al., 2010).

Fig. 3. (a) HPLC chromatogram of S. cumini (L.) leaf extract (280 nm) (inset: HPLC spectrum of standards (50 μg/mL); (1) Gallic acid; (2) Catechin; (3) Epicatechin; (4) Caffeic acid; (5) p-coumaric acid; (6) Ellagic acid; (7) Quercetin; (b) Chromatogram of S. cumini (L.) leaf extract for Myricetin 3-O-rhamnoside (256 nm) (inset: Myricetin 3-O-rhamnoside standard at 50 μg/mL); (c) Chromatogram of S. cumini (L.) leaf extract for Ferulic acid (321 nm) (inset: Ferulic acid standard at 50 μg/mL). 50

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3.3. Identification and quantification of phenolic compounds

about 74% of the total phenolics were identified.

Figs. 3(a–c) showed the HPLC chromatogram of optimally obtained aqueous jamun leaf extract. The major phenolic acid in leaf extract was tannic acid (915 ± 27.45 μg/mL) followed by gallic acid (24.6 ± 0.73 μg/mL). The most dominant hydroxycinnamic acid in extract was caffeic acid (8.74 ± 0.26 μg/mL) followed by ferulic acid (3.28 ± 0.09 μg/mL) and p-coumaric (1.33 ± 0.04 μg/mL). The hexahydroxydiphenic acid in extract was ellagic acid (1.63 ± 0.05 μg/ mL). The two flavan-3-ols identified in the extract were catechin (31.2 ± 0.93 μg/mL) and epicatechin (0.83 ± 0.02 μg/mL). Two flavonols namely quercetin (6.05 ± 0.18 μg/mL) and myricetin 3-Orhamnoside (20.7 ± 0.62 μg/mL) were detected in the extract, and the most abundant compound was myricetin 3-O-rhamnoside. Thus, total phenolics in the jamun leaf extract was determined using the Folin–Ciocalteu assay as (1370 ± 41.74) μg GAE /mL while sum of the individual phenolic compounds of leaf extract was calculated as (1013.5 ± 30.4) μg GAE /mL demonstrating that the TPC determined by Folin–Ciocalteu analysis gave close results to sum of individual phenolic compounds. Furthermore, the results of the present study indicated that jamun leaf extract was rich in tannic acid (67% of TPC) and

3.4. Extraction kinetics From the engineering point of view and better understanding of the process, selection of appropriate kinetic equation was essential to model the solid-to-liquid aqueous extraction of total polyphenols from jamun leaves. In order to investigate the aqueous extraction kinetics of jamun leaf, the amount of TPC extracted per 100 g of leaf powder was plotted as a function of extraction time for various extraction temperatures (34.8 °C, 60 °C, 85.2 °C) and L/S ratios (9.8:1, 35:1, 60.2:1 mL/g) as shown in Fig.4a. It was worth highlighting the presence of two distinct periods irrespective of the combination of process parameters: a rapid increase in the concentration of TPC at the beginning of the process and then showed a slow increase before reaching the equilibrium TPC concentration in aqueous extract. The optimal extraction condition could also be determined from the extraction kinetic data because of certain physical basis of each kinetic model. It could be noticed that for all the extraction conditions, TPC yield remained almost invariant beyond 60 min of operation (Fig. 4a). Thus, 60 min seemed to be optimal duration for extraction process. Among all the

Fig. 4. (a) Aqueous extraction of total polyphenols from jamun leaf for various operating conditions (Error bar: ± 3%); (b) Arrhenius plot of aqueous extraction of total polyphenols from jamun leaves. ■Pseudo-first order model (R2 = 0.96), y = - 2.341 + 1.143x; ● Minchev and Minkov model (R2 = 0.95), y = 2.191 + 1.10x; ▲Pseudo-second order model (R2 = 0.73), y = 1.792 + 0.1626x; ▭Peleg’s model (R2 = 0.92), y = 6.314-1.743x; (c) Pseudo-first order kinetic plot of the aqueous extraction of jamun leaves with L/S ratio of 35:1 and temperature of 60 °C. 51

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tested conditions, at the end of 90 min operation, the maximum TPC yield (6.2 g GAE/100 g leaf) was obtained at the liquid-to-solid ratio of 60.2 mL/g and temperature of 60 °C. At liquid-to-solid ratio of 35 mL/g and extraction temperature of 60 °C, at the end of 90 min operation, the obtained TPC yield was 5.8 g GAE/100 g leaf, which was about 94% of the maximum achievable yield. However, at higher temperature (85.2 °C), the TPC yield was obtained as 5.6 g GAE/100 g leaf. Therefore, the zone of optimal extraction conditions for obtaining maximum TPC yield depicted the liquid-to-solid ratio to be in the range of 35 mL/ g and 60.2 mL/g and extraction temperature close to 60 °C. This observation was corroborated with the optimal results obtained from RSM analysis (42.7 mL/g, 68.3 °C and 61 min) with TPC yield about 5.85 g GAE/100 g leaf. Thus, one could obtain the same interpretation from kinetic data regarding optimal extraction conditions derived from RSM analysis. Experimental kinetic data were elaborated using two most frequently used empirical models including Peleg’s model (Peleg, 1988) and Minchev and Minkov model (Minchev and Minkov, 1984) and two physical models including pseudo-first order model (Khan et al., 2010) and pseudo-second order model (Qu et al., 2010) reported in the literature to model kinetics of extraction of TPC from plant materials. The goodness of the various kinetic models fit to the experimental extraction data was established by the linear correlation coefficient (R2), the root mean square deviation (RMSD) and percent error. The results shown in Table 1 implied that extraction rate constant increased with increase of both temperature and L/S ratio for all the models. This further indicated that the extraction rate increased when the temperature or L/S ratio rose. However, rate constant was found to be more sensitive to temperature rather than L/S ratio. As an example, k-values estimated by pseudo-first order model increased from 0.246 to 0.409 min−1 with increase in temperature from 34.8 to 85.2ºC for fixed L/S ratio of 35 mL/g, while, k-values increased from 0.324 to 0.451 min−1 with increase in L/S ratio from 9.8 to 60.2 mL/g for a fixed temperature of 60 °C. Comparing the results of R2, RMSD and percent error (Table 1), all the four models tested were found to be suitable to

represent the extraction kinetics of total polyphenols from jamun leaves in the range of operating conditions studied. The high values of correlation coefficient (R2 > 0.96), low values of RMSD (< 0.5), and percent error (< 1.5%) indicated good concordance between experimental and calculated data. Thus, estimation of error was not enough to select the best model among them. Apart from modeling concentration of total phenolic content extracted as a function of time, another criteria generally used to evaluate how well models represent the experimental data was the relationship between extraction rate constant and extraction temperature. For solid-to-liquid extraction process, Arrhenius model was generally used to explain the temperature dependence extraction rate constant followed by determination of activation energy (Ea) using Arrhenius equation (Eq.2). The activation energy for total phenol extraction was determined from the slope of the plot of ln k against 1/T as observed in Fig.4b. It was observed that extraction rate constant obtained from both the empirical models, namely Peleg’s model and Minchev and Minkov model showed the temperature dependence according to Arrhenius equation with high R2 value of 0.92 and 0.95, respectively, and activation energy of 14.49 kJ mol−1 and 9.15 kJ mol−1, respectively for the tested temperature range. These models were found comparable with that of physical model, pseudofirst order model (R2 = 0.96 and Ea = 9.5 kJ mol−1). However, rate constant obtained from pseudo-second order model yielded lowest value of R2 (0.73) and activation energy of 1.35 kJ mol−1 and hence, pseudo-second order model was not considered good. Peleg’s model and Minchev and Minkov model, both were empirical in nature. However, pseudo-first order rate equation was derived from Fick’s second law at steady-state condition (Spiro et al., 1989). The knowledge of the relationship between extraction rate constant and temperature was a very important. Use of physical model was more appropriate for the purpose of design, process optimization and scale-up. Keeping in view the above facts, the pseudo-first order model appeared to be more appropriate and was selected as the best model to describe the aqueous extraction of total polyphenols from jamun leaves. Fig.4c showed the plot of ln[C /(C -Ct ) ] against extraction time for various L/S ratios according to pseudo-first order rate equation. As shown in Fig.4c, the extraction of phenolic compounds was divided distinctly into two regions: (i) the short-term extraction due to rapid washing of polyphenols from the superficial sites of plant material represented by a straight line with a relatively steep slope and (ii) the long-term extraction due to slow diffusion of polyphenols from internal sites of the plant materials to the bulk of the liquid extract represented by a straight line with a relatively gradual slope. The intersection between the washing stage and diffusion stage was the transition point indicating the changes of the extraction phase from the washing step to the diffusion step (Kandiah and Spiro, 1990). As observed in Fig.4c, the time required for onset of diffusion step was about 8 min, at the extraction temperature of 60 °C and L/S ratio of 35:1. Furthermore, washing stage led to extract 86% of total polyphenols extracted under the condition employed.

Table 1 : Kinetic and statistical parameters of various models for aqueous extraction of TPC from jamun leaves. Models

L/S Ratio (mL/g) Temp. (°C)

9.8 60

35 60

60.2 60

35 85

35 34.8

Minchev and Minkov Ct = A-B exp (-kt)

A B k R2 RMSD Error (%) C∝ k R2 RMSD Error (%) C∝ k R2 RMSD Error (%) k1 k2 R2 RMSD Error (%)

2.638 2.547 0.308 0.968 0.139 0.78 2.633 0.324 0.966 0.145 0.68 2.849 0.189 0.998 0.055 −1.43 0.681 0.355 0.996 0.04 0.091

5.455 5.380 0.351 0.982 0.221 0.36 5.456 0.357 0.982 0.224 0.30 5.865 0.0996 0.998 0.117 −0.96 0.294 0.172 0.994 0.11 −0.17

5.808 5.672 0.437 0.962 0.338 0.26 5.804 0.451 0.961 0.343 0.19 6.273 0.1105 0.998 0.154 −0.40 0207 0.163 0.994 0.13 0.021

5.230 5.098 0.396 0.964 0.294 0.37 5.226 0.409 0.964 0.300 0.31 5.672 0.107 0.998 0.138 −0.31 0.258 0.180 0.994 0.11 0.045

4.540 4.491 0.243 0.988 0.148 0.28 4.538 0.246 0.988 0.150 0.34 4.810 0.0992 0.998 0.209 −3.88 0.557 0.204 0.988 0.15 −0.74

Pseudo-first order Ct= C∝ (1-exp (-kt)) Pseudo-second order

Ct =C2 kt/(1+ C∝ kt)

Peleg's model Ct = t/(k1+k2t)

3.5. Kinetic compensation The value of extraction rate constants and correlation coefficient (R2), obtained from fitting of pseudo-first order equation for each L/S ratio and temperature value during extraction of polyphenols from jamun leaves, were presented in Table 2. It was clear from the table that extraction rate constant (k) increased with temperature as well as L/S ratio. For example, the rate constant (k) varied from 0.246 to 0.409 min−1 for temperature range of 34.8–85 °C at a fixed L/S i.e., 35 mL/g. Table 2 also indicated that all the R2 values > 0.95, confirmed the validation of experimental data. The activation energy for TPC extraction was calculated using Arrhenius equation (Eq.2). Fig.5(a) showed the variation of the frequency factor (lnk0) with activation energy (Ea) under different L/S ratio values. The linear behavior of the plot (R2 = 0.94) indicated the existence of compensation. It was worth to mention from Fig. 5(a) that an increase in the L/S ratio value from

Ct: Concentration of TPC at time t, g GAE/ 100 g leaf; C∝: Concentration of TPC at saturation, g GAE/ 100 g leaf; k: Extraction rate constant, min−1; k1: Peleg’s rate constant, 100 g leaf. min/g GAE; k2: Peleg’s capacity constant, 100 g leaf/g GAE.

exp cal Error(%) = [(C exp t -Ct )/Ct ] × 100 and RMSD(%) =

[(

N 1

exp 2 cal [(Cexp t -Ct )/Ct ] ) /(N -1)] .

52

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Table 2 Variation of extraction rate constant for different values of L/S ratio and temperatures. L/S ratio (mL/g)

Temp. (°C)

k (min−1)

R2

9.8

34.8 60 85 34.8 60 85 34.8 60 85

0.246 0.324 0.390 0.246 0.357 0.409 0.320 0.451 0.552

0.965 0.966 0.966 0.988 0.982 0.985 0.988 0.961 0.964

35 60.2

Table 3 Equilibrium parameters and Gibbs free energy of activation obtained from the pseudo-first order kinetic model for different values of L/S ratios and temperature. L/S ratio (mL/g)

Temp. (ºC)

keq x1016 (min−1)

ΔG#x10−3 (J/mol)

9.8

34.8 60 85 34.8 60 85 34.8 60 85

6.38 7.79 8.72 6.39 8.57 9.14 8.31 10.8 12.3

89.59 96.31 103.2 89.59 96.04 103.1 88.91 95.40 102.2

35 60.2

9.8 to 60.2 for the same range of temperatures caused new activation energy 1.17 times higher, while the new frequency factor value increased by 1.45 times. This implied that the increase in the rate constant by increasing the L/S ratio value was due mainly to a considerable increase in the frequency factor (number of collisions between molecules) rather than the change in the activation energy. The existence of real compensation effect was further examined by comparing the Tiso with the Thm. From the slope and intercept of this plot, the isokinetic temperature (Tisokin) and rate constant (kisokin) were calculated to be (-56.5 ± 7.9)ºC, and (5.8 ± 0.79)x10-2 min−1, respectively. For the given temperature range (34.8-85ºC), Thm was found to be 58.7ºC which was higher than Tisokin. Also, Thm did not lie within the complete confidence interval for the Tisokin (from −49 to -64.8 °C) and thus the compensation could not be due to propagation of experimental error. Therefore, kinetic compensation could be concluded to be real and the polyphenol extraction was entropic controlled. Fig.5(b) showed the kinetic constants of the polyphenol extraction at each L/S ratio and temperature. This figure clearly showed the isokinetic point (-56.5ºC, 5.8 × 10-2 min−1) which was the common intersection point for the entire kinetic data, further indicated that the compensation was real.

slope and the activation entropy (ΔS#) from the intercept. Activation enthalpy and entropy varied simultaneously to confirm the compensation thermodynamically under different L/S ratio (Fig. 6(a)). The coefficient of determination (R2 = 0.96) further concluded the linearization effect between enthalpy and entropy. The parameters p and q of Eq.(8) were obtained by linear regression from the data shown in Fig.6(a), giving the iso-equilibrium temperature (Tisoeq) and rate constant (kisoeq) as (-59.8 ± 7.0)ºC, and (2.33 ± 0.24)x10−16 min-1, respectively. Any change in the L/S ratio caused higher changes in the new activation entropy (ΔS#) value and thus the extraction was more sensitive to changes in entropy rather than changes in the temperature. At iso-equilibrium temperature, the free energy of activation was found to be 63.80 kJ/mol, indicating that if the extraction could be done at the iso-equilibrium temperature with any L/S ratio value within the studied range, the free energy of activation would be the same, regardless of the L/S ratio value. Fig.6(b) showed the variation of equilibrium constant (keq) with temperature at the different L/S ratio values investigated. There was an iso-equilibrium point (-59.8ºC, 2.33 × 10−16 min-1) in the plot for each L/S ratio value, which was common intersection point for the entire data, further indicated that the compensation was real. Also, Tisoeq was lower than the harmonic mean temperature (58.7 °C), the control could be concluded as entropic. Similar kind of observation was reported by Flores-andrade et al., (2009). The isokinetic temperature obtained using thermodynamic compensation (-59.8ºC) was very close to that obtained using kinetic compensation (-56.5ºC), confirming the existence of both compensation processes for the extraction of TPC from jamun leaves.

3.6. Thermodynamic compensation Table 3 showed the values of equilibrium constant (keq) and Gibbs free energy of activation (ΔG#) obtained from Eq.(6) and Eq.(9), respectively, at each value of L/S ratio and temperature during extraction of polyphenols from jamun leaves. Linear regression analysis of ln(keq) vs 1/T allowed the estimation of activation enthalpy (ΔH#) from the

Fig. 5. (a) Linear relationship between lnk0 and Ea; (b) kinetic constants of the polyphenol extraction at each L/S ratio and temperature. 53

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Fig. 6. a) Linear relationship between activation enthalpy (ΔH#) and entropy (ΔS#); (b) Equilibrium kinetic constants of the polyphenol extraction at each L/S ratio and temperature.

It was worth to mention that for the study of thermodynamic compensation, the equilibrium constant was obtained from the extraction rate constants assuming the transition state theory and other

equilibrium parameters ( G# , H# , and S# ) were obtained for each L/S ratio by fitting to the Van't Hoff equation (Eq.7). The thermodynamic compensation referred solely to equilibrium stage involved in the

Fig. 7. Prediction of permeate flux by (a) complete blocking model, (b) intermediate pore blocking, (c) standard pore blocking, (d) cake filtration model, for MF with different membrane pore sizes at stirrer speed of 1000 rpm and transmembrane pressure of 138 kPa (lines: predicted data; symbols: experimental results). 54

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transition state. If the extraction process was not assumed to follow the transition state theory, the calculation of all the equilibrium parameters would not make any sense. The study of thermodynamic compensation

could be misleading, because the equilibrium parameters would not be correctly related to the complete extraction process. Therefore, the true equilibrium parameters for the extraction process could not be obtained

Fig. 8. Effects of MF membrane pore sizes on (a) variation of relative flux with time (b) absolute permeate flux and volume reduction ratio (c) AIS removal and extract clarity (d) extract purity and recovery of TPC and TFC. The error bars represent standard deviation for n = 3; Pictorial representation of (e) jamun leaf powder, and jamun leaf extract and microfiltered extract during storage at 4ºC (1 and 1′: leaf extract for 0 day and 45 days, respectively; 2 and 2′: 0.1 μm microfiltered extract for 0 day and 45 days, respectively; 3 and 3′: 0.22 μm microfiltered extract for 0 day and 45 days, respectively; 4 and 4′: 0.45 μm microfiltered extract for 0 day and 45days, respectively; 0.8 μm microfiltered extract for 0 day and 45 days, respectively). 55

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(Garvín et al., 2017). It could also be mentioned that thermodynamic compensation always occurred if the kinetic compensation also took place because the equilibrium constant and the extraction rate constant were related through the transition state theory. On the other hand, if the experimentally obtained equilibrium constants data were fitted to the Van't Hoff equation so as to obtain the other equilibrium parameters ( G , H , and S ) for each L/S ratio, only the thermodynamic compensation could be studied. In this case the kinetic compensation would not make any sense although the extraction rate constants could be calculated mathematically from Eq.6. Thus, one could say that the kinetic compensation was beneficial and thermodynamic compensation was more desirable as far as the practical benefit of the process.

while AIS removal efficiency decreased. For 0.1 μm membrane, most of the larger molecular weight solutes and suspended solids were rejected due to smaller pore size of the membranes and dynamic cake type layer formed over the membrane surface leading to an increase in AIS removal and permeate clarity. This deposited cake layer rejected some more polyphenols resulting in decrease in the recovery of both TPC and TFC with low value of absolute permeate flux of 20.6 L/m2 h obtained at the 60 min. of operation with VRR of 31.4% (Fig.8b). On the other hand, for higher pore size membrane (0.8 μm), at the very beginning of experiment (within 30–60 sec of the experiment), some of the larger molecular weight polysaccharides and suspended solids along with polyphenols passed through the membrane with a very high initial permeate flux leading to decrease in AIS removal efficiency (only 6.8%) and permeate clarity (64.4%). Due to passage of more polyphenols, the recovery of both TPC and TFC in the permeate increased. Fig.8a showed the normalized permeate flux as a function of operating time for MF with different membrane pore sizes. Interestingly, as observed in Fig.8a, the 0.8 μm membrane having highest permeability, showed the steepest flux decline (less than 1% of the initial flux) with highest flux decline ratio and yielded the lowest absolute permeate flux of 7.8 L/m2 h at the 60 min. of operation among all the tested membranes. This might be explained by the fact that, due to larger pore size, more solutes can enter the pores and cause them to become blocked rapidly resulting to a severe concentration polarization and cake formation. However, due to more AIS content in the permeate, purity of clarified extract decreased. For 0.8 μm membrane, the lower extract purity of 30% in the clarified extract could be possibly explained by increasing the permeation of larger molecular weight solutes in the permeate (high AIS content) due to its larger pore size. Also 0.1 μm membrane yielded extract with low purity of 31%, which might be due to low recovery of TPC of 56% in the permeate as the purity depended on relative ratio of phenolic and non-phenolic compounds in the extract (Fig.8b). Therefore, 0.8 and 0.1 μm membranes were not found to be suitable for clarification of jamun leaf extract. Significant VRR of about 65.7% was observed in case of microfiltration using 0.45 μm membrane. The absolute permeate flux at 60 min. of operation and AIS removal efficiency were determined as 29.6 L/m2 h and 61%, respectively for 0.45 μm membrane and 23.7 L/m2 h and 66%, respectively for 0.22 μm membrane. The highest absolute permeate flux and maximum extract purity (35%) corresponded to clarified extract obtained with 0.45 μm membrane, with VRR of 65.7%. This is consistent with the results of minimum fouling coefficients obtained for 0.45 μm membrane. The AIS removal efficiency and extract clarity obtained with 0.22 and 0.45 μm membranes were comparable. Furthermore, about 72% and 61.2% of TPC permeated through 0.45 and 0.22 μm membranes, respectively. Similar results were obtained by Liu et al., (2013), during stirred deadend microfiltration of grape pomace extracts using 0.2 μm membrane, and achieved a recovery of total phenolic content of about 60%. The extract clarity was found to be greater than 80% with all membranes except 0.8 μm membrane. Although extract purity was not improved

3.7. Microfiltration of jamun leaf extracts 3.7.1. Fouling mechanism The aqueous jamun leaf extracts obtained at optimized conditions (temperature: 68.3 °C, L/S ratio: 42.7:1, and time: 61 min) was used as the feed for clarification process with MF membranes of different pore sizes. Figs.7(a–d) showed the fitting of the experimental flux data obtained in microfiltration of jamun leaves extract to the Hermia’s model for various fouling mechanisms. It was noticed that the decline in flux was very rapid at the beginning of experiment and then continued to decrease gradually with time. This might be explained by the fact that in the batch concentration mode of operation, permeate was not recycled back to the feed tank and therefore, the solute concentration in the retentate kept on increasing, leading to gradual increase in concentration polarization and cake type layer formation over the membrane surface. This resulted to a gradual decrease in permeate flux with time. This behavior was similar for all the tested membranes. The appropriate fitness and capability to measure the various fouling mechanisms could be confirmed by comparing the coefficient of determination (R2) obtained from the linear regression analysis. Cake filtration mechanism had the highest R2 values (0.86-0.98) while complete pore blocking mechanism had the worst R2 values (0.25-0.88). The fouling mechanism during filtration was generally depended on the membrane material, pore size, operating conditions, and nature of solutes to be filtered etc. In the present study, for all the membranes tested under a fixed operating conditions, the best fit to experimental data corresponded to the cake layer formation model followed by the intermediate pore blocking model, with values of R2 ranging from 0.86 to 0.98 in the case of the cake layer formation model and ranging from 0.67 to 0.97 for the intermediate pore blocking model. The flux decline was attribute to the pore blocking followed by gradual growth of cake type layer, constituted by tissues, suspended materials, cell debris, large polyphenols etc. over the membrane surface. This might be because the size of the components in jamun leaves extract were either comparable or larger than the pore size of these membranes. It was observed that for fixed values of stirrer speed and transmembrane pressure (1000 rpm and 138 kPa), the cake filtration fouling coefficients, kc, were 7.65 × 106, 8.19 × 106, 3.62 × 106, and 64.5x x106 s/m2 for 0.1, 0.22, 0.45, and 0.8 μm membranes, respectively. According to the definitions and physical meaning of Hermia’s model parameters, higher values of model parameters corresponded to more severe membrane fouling. Lowest kc value of 0.45 μm membrane indicated that 0.45 μm membrane offered less fouling during clarification process among all the tested membranes.

Table 4 Model parameters (K), and coefficient of determination (R2) for degradation of TPC and TFC in jamun leaf extracts during storage of six weeks (mean ± CI (95% confidence interval)). Extract

TPC

TFC 3

K*10 (week

3.7.2. Influence of membrane pore size on the permeate flux and permeate quality The performance of microfiltration process was evaluated according to the absolute permeate flux, recovery of TPC and TFC, VRR, AIS reduction and extract clarity as observed in Figs.8(a–d). The membrane pore size was found to have significant effect on both permeate flux and extract quality. With increase in the membrane pore size, VRR increased with increase in recovery of both TPC and TFC in the permeate

Original Extract Microfiltered Extract 0.1 μm 0.22 μm 0.45 μm 0.8 μm

56

−1

)

R

2

K*103 (week−1)

R2

30.2 ± 0.8

0.98

21.5 ± 0.85

0.96

9.8 ± 0.7 22.9 ± 1.4 25.4 ± 0.4 28.2 ± 0.5

0.93 0.94 0.99 0.98

7.5 ± 0.36 10.5 ± 0.5 11.7 ± 0.3 18.5 ± 0.25

0.95 0.95 0.98 0.99

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significantly after microfiltration, the clarification of leaf extract was necessary in removing suspended solids which might reduce the fouling phenomena in the following concentration step using nanofiltration or low cut-off UF membrane, leading to enhancement in process throughput. Based on the above findings, the membrane of 0.45 μm should be selected as the best membrane for clarification of jamun leaf extract among the all the membranes tested, since it yielded maximum absolute permeate flux with maximum extract purity and also enabled significant recovery of polyphenols (TPC and TFC) and removal of AIS with 56%.

4. Conclusions The aqueous extraction of phenolic compounds from jamun leaves was optimized using RSM. The optimal extraction conditions (extraction time, 61 min; extraction temperature, 68.3 °C; and L/S ratio, 42.7:1) showed higher yield (20.9%) and purity (28.0%) compared to those obtained from the other plant leaves. Tannic acid was the dominant among the six phenolic acids identified in the extract. The four flavonoid compounds identified were catechin, epicatechin, quercetin and myricetin 3-O-rhamnoside in the extract. Furthermore, about 74% of the total phenolics was identified in the present study. The pseudo-first order kinetic model was selected as the best model for describing the polyphenol extraction kinetics. The kinetic and thermodynamic compensations were found to occur for extraction of TPC from jamun leaves with the isokinetic and isoequilibrium temperatures of (-56.5 ± 7.9)ºC and (-59.8 ± 7.0)ºC, respectively. Both the compensations were concluded to be real and the extraction process, entropic. It further confirmed that the extraction rate was dependent on the L/S ratio value for the range studied. The potential of microfiltration process for the clarification of liquid extract was investigated to obtain a stable phenolic product with improved clarity and purity. Analysis of the flux decline data revealed that microfiltration of jamun leaf extract was controlled by a cake filtration mechanism. The maximum values of permeate flux (29.6 L/m2 h) and extract purity (35%) were achieved with the 0.45 μm membrane during clarification among the all the tested MF membranes. During 42 days storage at 4 °C, MFclarified leaf extract led to decrease in loss of TPC, TFC and color in comparison with the original leaf extract. The degradation of TPC and TFC of jamun leaf extract followed first order kinetics with respect to storage time. The obtained results clearly showed that the jamun leaf is a good source for producing phenolic compounds. Extraction coupled with microfiltration process is a viable technique for producing jamun leaf extract of desirable quality parameters with enhanced purity, clarity and storage stability.

3.8. Storage stability of leaf extracts To monitor the storage stability of extracts, the important quality parameters of both the leaf extracts such as TPC, TFC, color and clarity were determined initially and after each consecutive week up to 45 days. The initial value of TPC and TFC were 1380 mg/L and 133 mg/L in the original leaf extract while in the microfiltered extracts with 0.1, 0.22, 0.45, and 0.8 μm membranes, the initial values were 820, 896 1050, and 1250 mg/L, respectively, for TPC and 85, 89, 94, and 128 mg/L, respectively, for TFC. It was important to observe that the losses in TPC and TFC of microfiltered leaf extracts were lower than those found in original leaf extracts. During 45 days of storage, TPC in the jamun leaf extracts decreased in approximately 16%, while TPC in the microfiltered extract decreased to about 5.5%, 14%, 14% and 15% of the initial value, for the membranes of 0.1, 0.22, 0.45 and 0.8 μm, respectively. On the other hand, during the same storage period, the losses in the TFC of microfiltered extracts were 4.8%, 6.4%, 6.7% and 11.0% of the initial value, for the membranes of 0.1, 0.22, 0.45 and 0.8 μm, respectively, compared to 12% loss of TFC in case of original extracts. The degradation of TPC and TFC of jamun leaf extract followed first order kinetics with respect to storage time as, At = A 0exp(- Kt) , where A0 was the initial concentration of TPC/TFC (mg/L) and At was the concentration after time t (weeks) of storage at the 4 °C while K (week −1) was the first order degradation rate constant. The model was observed to properly match the experimental degradation data of both TPC and TFC with high coefficients of determination (R2 = 0.93–0.99). The degradation rate constants of TPC and TFC during storage were shown in Table 4. Microfiltration was found to have significant effect on the degradation of both TPC and TFC during storage. In fact, storage of original extract at 4 °C resulted in a much faster degradation compared with microfiltered extract. For example, the k values of TPC degradation were 30.2 × 10-3 week−1 for original extract, and 9.8, 22.9, 25.4, and 28.2 × 10-3 week−1 for microfiltered extracts with 0.1, 0.22, 0.45, and 0.8 μm membrane, respectively. The k values of TFC degradation were 21.5 × 10-3 week−1 for crude extract, and 7.5, 10.5, 11.7, and 18.5 × 10-3 week−1 for microfiltered extracts with 0.1, 0.22, 0.45, and 0.8 μm membrane, respectively. Photographs of jamun leaf powder, original leaf extracts and MF-clarified extracts obtained with different pore size of MF membrane at various storage times were shown in Fig.8e. Degradation of phenolic compounds leading to development of brownish color during storage was more pronounced in case of original leaf extract when compared to the MF-clarified extracts stored at 4 °C. Color analysis also revealed an increase in the absorbance at 420 nm with storage time. Furthermore, the reduction in the clarity (15%) of the leaf extracts at the end of the storage at 4 °C was higher than the reduction in the clarity (only 3%) of the MF-clarified extracts obtained with the membrane of pore size 0.45 μm. However, no microbial growth was visually observed during 45 days of storage for all the extracts. A slight decrease in antioxidant activity of MF-clarified extract of 5–10% was observed during 45 days of storage (data not shown) which might be related to the observed losses of phenolic compounds. These results also confirmed the greater stability of the MF-clarified extracts.

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