Purification of anthocyanins from jamun (Syzygium cumini L.) employing adsorption

Separation and Purification Technology 125 (2014) 170–178

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Purification of anthocyanins from jamun (Syzygium cumini L.) employing adsorption Chandrasekhar Jampani, Aduja Naik, K.S.M.S. Raghavarao ⇑ Department of Food Engineering, CSIR – Central Food Technological Research Institute (CFTRI), Mysore 570020, India

a r t i c l e

i n f o

Article history: Received 9 May 2013 Received in revised form 3 January 2014 Accepted 27 January 2014 Available online 7 February 2014 Keywords: Adsorption Anthocyanins Syzygium cumini L. Non-enzymatic browning Adsorption isotherms

a b s t r a c t The present study deals with the downstream processing of anthocyanins from jamun (Syzygium cumini L.) in order to obtain anthocyanins in a purified form. Adsorption was carried out employing six different adsorbents and among these, Amberlite XAD7HP showed the highest adsorption capacity (1.07 mg/mL of adsorbent) and desorption ratio (87.62%). Aqueous acidified ethanol (above 40%, v/v) could effectively elute the anthocyanins. Adsorption results were found to correlate best using the Langmuir equation at all the temperatures studied. Second order kinetics model was found to be more appropriate to describe the adsorption of anthocyanins. The dynamic adsorption process parameters for the purification of anthocyanins using Amberlite XAD7HP arrived at were as follows; for adsorption: processing volume, flow rate and temperature were 6.5 BV, 1 mL/min and 25 ± 1 °C, respectively and for desorption: eluent volume and flow rate were 4 BV of acidified aqueous ethanol (40%, v/v) and 1 mL/min, respectively. Anthocyanins extract after purification was found to be free of sugars, which are the major cause for degradation of anthocyanins. After the purification by adsorption, the degradation constant and non-enzymatic browning index of anthocyanins were found to decrease from 0.93 to 0.19 and 0.45 to 0.29, respectively. The qualitative determination of anthocyanins after adsorption was evaluated by the physio-chemical characteristics and the structural stability was confirmed by HPLC–MS/MS. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Recently, research on the natural colors has increased because of their possible health benefits over synthetic food colorants. In this regard, anthocyanins being natural colorants, attention on the purification of these pigments has been on the rise [1,2]. Anthocyanins are a group of water-soluble flavonoids that are responsible for the bright red, blue and purple colors in fruits, vegetables and ornamental crops. The anthocyanins found in foods are glycosides of six main aglycon anthocyanidins: cyanidin, delphinidin, pelargonidin, peonidin, malvidin and petunidin. Health benefits associated with the use of anthocyanin-enriched (colored) foods include reduced risk of coronary heart disease [3], protection against obesity and hypoglycemia [4], memory enhancement [5] and prevention of cancer [6]. There are a good number of sources rich in the anthocyanins content, jamun (Jambolao) being one among them. The total anthocyanin content of the jamun is higher than that in other fruits such as Acerola, Camu-camu and Jaboticaba [7,8]. Eugenia jambolana Lam. (Syn. Syzygium cumini Skeels or Syzygium jambolana Dc or Eugenia cuminii Druce.) belonging to ⇑ Corresponding author. Tel./fax: +91 821 2513910. E-mail address: [email protected] (K.S.M.S. Raghavarao). http://dx.doi.org/10.1016/j.seppur.2014.01.047 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.

the family Myrtaceae is a large evergreen tree indigenous to the Indian subcontinent [9]. However, today these trees are found growing throughout the Asian subcontinent, Eastern Africa, South America, Madagascar and have also naturalized to the warmer regions of the United States of America [10]. The fruits are oblong berries, deep purple or bluish in colour with pinkish pulp, having various medicinal properties such as stomachic, astringent, antiscorbutic, diuretic, antidiabetic, antioxidant, antiproliferative and efficacy in reducing the risk of enlargement of spleen [11–14]. The fruit concentrate of jamun has a very long history of use for various medicinal purposes and currently has a large market for the treatment of chronic diarrhoea and other enteric disorders [15]. Jamun fruit extract showed antiproliferative and pro-apoptotic effects against breast cancer cells [16]. Extraction methods of anthocyanins from plant material are non-selective and yield pigment solutions with large amounts of other compounds such as sugars, sugar alcohols, organic acids, amino acids and proteins which are detrimental to stability of pigments. Stability of colorants is strongly influenced by sugars, light, oxygen, pH, temperature and UV-light [17]. The free sugars and their degradation products in the anthocyanins extract lead to the milliard reaction and form brown compounds. Hence, removal of sugars from anthocyanins extract is very much desirable for the

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stability of these pigments and also to facilitate their application in food processing. Adsorption is an effective method for purification of bioactive components in a single step [18,19]. Different adsorbents were reported for the separation of anthocyanins from crude extracts of cultivars of mulberry [20], Arona melanocarpa var Nero [21] and red cabbage [22–24]. In-spite of a large number of research reports being available in the literature on enormous medicinal and food applications, there is a paucity of information regarding the purification of jamun anthocyanins by any method especially by adsorption. Hence, the objective of the present work is a detailed study of the purification of jamun anthocyanins employing adsorption followed by evaluation of colorant properties after the adsorption.

where C0 and Ce are the initial and equilibrium concentrations of anthocyanins in the solution (mg/mL), respectively. Vi and Va are the volumes of initial sample solution and adsorbent, respectively. Desorption was performed as follows: After ensuring the adsorption equilibrium is reached, resins were washed by deionized water and then desorbed with 40 mL of ethanol (0.1% v/v HCl). The eluent samples were analyzed by spectrophotometer. The following equations were used to quantify the desorption capacity (qd – mg/mL of resin) at desorption equilibrium and ratio of desorption (D, %)

qd ¼

CdV d Va

ð3Þ

2. Materials and methods

D¼

Cd V d  100 ðC 0  C e ÞV i

ð4Þ

2.1. Materials

where Cd is the concentration of the solute in the eluent solution (mg/mL); Vd is the volume of the eluent (mL). C0, Ce, Vi and Va are same as described earlier.

Adsorbents namely, Amberlite XAD7HP, Amberlite XAD4, Amberlite IRC 86, Amberlite IRC 120, Dowex 50xw8 (hydrogen form) and Silica gel (50–100 mesh) were obtained from Sigma Aldrich, St. Louis. Jamun fruits were purchased from local market. Potassium chloride, sodium acetate and sulphuric acid were obtained from Ranbaxy chemicals Ltd., Mumbai. L-Ascorbic acid was procured from Himedia Pvt. Ltd., Mumbai. All the chemicals used were of analytical grade. 2.2. Methods 2.2.1. Extraction of anthocyanins Seeds were removed from the jamun fruits and extraction was carried out by employing water as a bio-solvent while providing thorough contact in a mixing unit (Singer, India FP-450). The ratio of pulp to extraction medium (water) was maintained at 1:2. Ascorbic acid (0.1% w/v) was added to inhibit the activity of polyphenol oxidase. The anthocyanins extract obtained was filtered using a muslin cloth to remove the coarse particles. The filtrate obtained was centrifuged at 6000 rpm for 10 min to remove fine suspended particles. The anthocyanins concentration in the extract was estimated using pH differential method, employing the following equation [25]

Anthocyanins concentration ðmg=LÞ ¼

A  Mw  DF eL

ð1Þ

where A = [(A530–A700)pH 1.0 – (A530–A700)pH 4.5], Mw is the molecular weight of anthocyanins (449.2 g/mol), DF is the dilution factor, e is the extinction coefficient (26,900 L/cm mol) and L is the path length (1 cm). Absorbance at 530 and 700 nm was measured using UV–VIS spectrophotometer (Double beam spectrophotometer, Shimadzu, Japan, Model UV-160A). 2.2.2. Static adsorption and desorption tests The adsorption process of jamun anthocyanins on macroporous resins was carried out in the following manner. Activation of adsorbent was performed by overnight treatment with 2 bed volumes (BV) of distilled alcohol followed by rinsing with 5 BV of distilled water. All the adsorption experiments were carried out in batch mode at 25 ± 1 °C. 2 mL of each adsorbent (activated) was contacted with 40 mL of anthocyanins extract in 100 mL conical flask while agitating on a vibratory shaker. The solutions after adsorption were analyzed for anthocyanins. The following equation was used to quantify the adsorption capacity (qe – mg/mL of adsorbent) at equilibrium.

qe ¼

ðC 0  C e ÞV i Va

ð2Þ

2.2.3. Adsorption isotherms The correlation of equilibrium adsorption data by either theoretical or empirical equations is important for the standardization of adsorption systems. Equilibrium adsorption isotherm studies of the selected adsorbent were conducted by contacting 40 mL of anthocyanins extract of different concentrations with 2 mL of adsorbent for 50 min while agitating on a vibrator shaker at 5°, 15° and 25° ± 1 °C, respectively. The initial and equilibrium concentrations at these temperatures were determined by spectrophotometric method. In this study the Langmuir and Freundlich models were employed to describe the equilibrium adsorption [26,27]. The Langmuir isotherm is best known for monolayer adsorption and the equation is given by

qe ¼

qm C e K L þ Ce

ð5Þ

The above equation can be rearranged into a linear form with Ce and Ce/qe as independent variable as

Ce K L Ce ¼ þ qe qm qm

ð6Þ

where KL is the adsorption equilibrium constant and qm is the empirical constant. These constants can be obtained by plotting a graph between Ce/qe and Ce and corresponding regression coefficient (R2) values can be noted. The model widely used for non-ideal adsorptions is Freundlich isotherm, an empirical equation and can be expressed as

qe ¼ K F C 1=n e

ð7Þ

The above equation (Eq. (7)) can be rearranged as

log qe ¼ log K F þ

1 log C e n

ð8Þ

where KF is the Freundlich constant (adsorption capacity) and 1/n (adsorption intensity) is an empirical constant. These constants can be obtained by plotting a graph between log qe and log Ce and corresponding regression coefficient (R2) values can be noted. 2.2.4. Adsorption equilibrium and kinetics Activated adsorbent (2 mL) was contacted with 40 mL of anthocyanins extract of jamun in a 100 mL conical flask while agitating on a vibratory shaker. Aliquots of 1000 lL were drawn from the solution at regular intervals of 5 min, up to 60 min, in order to identify the time at which the adsorption process attains equilibrium. Analysis was carried out for these aliquots and the amount of anthocyanins adsorbed was calculated by the difference.

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Pseudo first-order [28] and pseudo second-order [29] kinetic models were used in the present work for the analysis of adsorption of anthocyanins on Amberlite XAD7HP. The conformity between experimental data and the values of adsorption capacity predicted by the model was expressed by the correlation coefficients (R2 values). A relatively high R2 value obviously indicates that the model successfully describes the kinetics of adsorption of anthocyanins. 2.2.4.1. First order kinetics. The Lagergren rate equation is one of the most widely used adsorption rate equations for the adsorption of solute from a solution. The pseudo-first-order kinetic model of Lagergren is generally expressed by

dqt ¼ k1 ðqe  qt Þ dt

ð9Þ

where qe and qt are the adsorption capacity (mg/mL), at equilibrium and at time t, respectively. k1 is the rate constant of pseudo first-order adsorption (min1). After integration and applying limits of integration, initial condition at t = 0, qt = 0 and at t = t, qt = qt. The integrated form of Eq. (9) becomes

logðqe  qt Þ ¼ log qe 

k1 t 2:303

2.2.4.2. Second order kinetics. The second-order kinetic model is expressed by

ð11Þ

where k2 is second order rate constant. Rearranging the terms in Eq. (11) gives

dqt ðqe  qt Þ2

¼ k2 dt

ð12Þ

After integration and applying conditions (initial condition at t = 0, qt = 0 and at t = t, qt = qt), the integrated form of Eq. (12) becomes

1 1 ¼ þ k2 t ðqe  qt Þ qe

 ln

Ct Co

 ¼ K d t

ð15Þ

where Co (mg/L) is the initial concentration of anthocyanins and Ct (mg/L) is the concentration of anthocyanins at a specified time ‘t’ (days). The degradation studies were carried out by measuring the anthocyanins concentration each day, up to 10 days, at room temperature (25 ± 1 °C).

2.2.8. Color analysis The color characteristics (CIE L, a, b) of anthocyanins before and after purification were measured using a colorimeter (Lab Scan XE, Hunter Lab, Virginia). The sample was placed in a Petri plate against the white background and CIE L, a, b values were measured in the transmission mode, using Illuminant D65 and 2° observer angle.

ð13Þ

Eq. (13) can be rearranged into a linear form as shown below in Eq. (14)

t 1 1 ¼ þ t qt k2 q2e qe

2.2.7. Determination of sugars, soluble solids, non-enzymatic browning index and degradation constant The Dubois method [30] was used for the estimation of total sugars present in the extract. Dextrose was used as a standard for the determination of sugars. Soluble solid content, expressed as refractive index, was measured using refractometer (ERMA, Japan). Browning index describing the quality of anthocyanins was estimated [31] as Browning index = absorbance at 420 nm of bisulfate treated sample Degradation constant (Kd) of anthocyanins at room temperature (25 ± 1 °C) was calculated considering first-order degradation kinetics as per the following equation [32]

ð10Þ

The values of qe and k1 can be obtained from the intercept and slope, respectively of the plot log(qe  qt) vs t.

dqt ¼ k2 ðqe  qt Þ2 dt

(BV) of the adsorbent was 20 mL and packed length of adsorbent was 11.2 cm. Anthocyanins extract was passed through the column at different flow rates (1, 2 and 3 mL/min). Fractions of 10 mL were collected and the concentration of anthocyanins was analyzed. After reaching the adsorptive saturation, the adsorbent in the column was washed thoroughly using deionized water (5 BV) followed by elution using aqueous ethanol (40% v/v) at different flow rates (1 and 2 mL/min).

ð14Þ

The equilibrium adsorption capacity (qe) and the second order constant (k2) can be determined from the slope and intercept, respectively of the plot t/qt vs t. 2.2.5. Elution experiments To determine the optimum ethanol concentration for elution of the adsorbed anthocyanins, 2 mL of Amberlite XAD7HP adsorbent saturated with anthocyanins was contacted with 50 mL of different proportions of acidified (1% HCl, v/v) aqueous ethanol (water:ethanol) in conical flasks while agitating using a vibratory shaker. The contents of anthocyanins in the eluates were measured spectrophotometrically as described earlier (Section 2.2.1). 2.2.6. Dynamic adsorption and desorption tests Dynamic adsorption and desorption experiments were carried out in a glass column (20 mm  120 mm) packed with 20 mL of the selected adsorbent (Amberlite XAD7HP). The bed volume

2.2.9. HPLC–MS/MS analysis of anthocyanins The HPLC analysis of anthocyanins was performed as per the procedure described by Veigas et al. [33]. The purified anthocyanins were separated using a C18 Shim-pack CLC–ODS column (5 lm, 250  4.6 mm i.d.), analysed by HPLC (Waters Alliance 2695 HPLC) equipped with an auto sampler and coupled with a photodiode array detector (Waters 2696) and a Q-TOF UltimaTM mass spectrometer, utilizing the electro spray ionization (ESI– MS) interface (Waters Corporation, Manchester, UK). The mobile phase (0.6 ml min1) consisted of (A) water (1% v/v formic acid) and (B) methanol (1% v/v formic acid). The gradient was: 0 min, 15% B; 0–20 min, 15–30% B; 20–25 min, 30–35% B; 25–35 min, 35–40% B; 35–42 min, 40% B; 42–43 min, 40–100% B; 43– 48 min, 100% B; and 48–49 min, 100–15% B, followed by equilibration for 5 min at 15% B. Chromatograms were acquired at 530 nm. Samples (20 lL) were analysed in duplicate. Positive ion spectra of the column eluate were recorded in the range of m/z 20–2000 at a scan rate of 2 s/cycle under the following conditions: collision energy 10.0; capillary voltage 35 V; cone voltage 100 kV; source temperature 30 °C; desolvation temperature 110 °C; cone gas flow 0.4 L/min; desolvation gas flow (8.3 L/min). Argon was used as the collision gas. Data acquisition and processing was performed using MassLynxTM 4.0 SP4 software (Micromass).

C. Jampani et al. / Separation and Purification Technology 125 (2014) 170–178

3. Results and discussion The extraction of anthocyanins was carried out using 500 g of jamun pulp and 1000 mL of extraction medium (water). The crude extract obtained (1100 mL) after the centrifugation was stored at 4 °C and used for further experiments. The results of adsorption experiments are discussed in the following sections. 3.1. Adsorption and desorption capacities Experiments of both adsorption and desorption were carried out for the evaluation of different adsorbents. The results are presented in Fig. 1. Mode of adsorption and surface area of the adsorbents are given in the Table 1. It can be observed from the figure that Amberlite XAD7HP has shown highest adsorption and desorption capacities compared to other adsorbents which can be attributed not only to its similar polarity with the anthocyanins, but also to its high surface area. From the table, it can be seen that Amberlite XAD7HP is the highest in polar nature along with high surface area. Adsorbents similar in polarity to solute was observed to exhibit better adsorption ability. Similar results were reported in case of red cabbage anthocyanins [22]. The recovery of anthocyanins (percentage) of different adsorbents were evaluated and results are given in Fig. 2. It can be seen from the figure that the recovery of anthocyanins was high in case of Amberlite XAD7HP compared to that of other adsorbents. The adsorption and desorption capacities of Amberlite XAD7HP are 1.07 and 0.94 mg/mL of resin, respectively. Hence, further experiments have been carried out using Amberlite XAD7HP as an adsorbent.

173

Table 1 Modes of adsorption and characterstics of different adsorbents. Adsorbent

Mode of adsorption

Silica gel Amberlite IRC 80 Amberlite IR 120 DOWEX 50WX8 Amberlite XAD4

Reversed phase, non-polar Weakly acidic anion exchanger Weakly acidic cation exchanger Strongly acidic cation exchanger Non-ionic acrylic ester resin moderate polarity (hydrophobic in nature) Non-ionic acrylic ester resin moderate polarity

Amberlite XAD7

Fig. 2. Recovery (%) of anthocyanins on different adsorbents.

3.2. Adsorption isotherms The distribution of anthocyanins between the liquid and solid phases is a measure of the time required to reach equilibrium in the adsorption process and is expressed by the Langmuir and Freundlich isotherms. Accordingly, equilibrium adsorption isotherms, constructed at different temperatures of 5, 15 and 25 ± 1 °C were analyzed. The initial anthocyanins concentration of the extracts were maintained as 24.6, 49.2, 73.8, 98.4 and 123.07 mg/L. The results are presented in Fig. 3. It can be seen from the figure that the adsorption capacity increased with an increase in the initial concentration and reached the saturation plateau at 123.07 mg/L. The highest adsorption capacity (qe  1.0 mg/mL of adsorbent) was observed at a temperature of 25 ± 1 °C. Hence, it can be inferred that temperature of 25 ± 1 °C and concentration of 123.07 mg/L are the optimized conditions for adsorption of jamun anthocyanins. The constants of both Langmuir and Freundlich equations, respectively calculated from the slope and intercepts of the plots

Fig. 3. Adsorption isotherms of anthocyanins on Amberlite XAD7HP.

given in Fig. 4 are presented in Table 2. It can be observed from the table that qm and R2 values decreased with a decrease in temperature. The highest qm and R2 values were obtained in case of Langmuir model at 25 ± 1 °C. In case of Langmuir equation, qm (1.04 mg/mL) was found nearly equal to the experimental adsorption capacity (1.07 mg/mL of adsorbent) observed at a temperature of 25 ± 1 °C. Hence, it can be inferred that the adsorption of anthocyanins is explained well by Langmuir isotherm. In case of Freundlich model, the values of adsorption intensity (n) were observed to be in the range 0 < n < 10, which indicate that the adsorption of anthocyanins to be favorable. However, the correlation coefficients are very much lower than unity. Hence, it can be inferred that the adsorption of anthocyanins can be correlated best using the Langmuir equation especially at 25 ± 1 °C. Similar results were reported in case of adsorption of anthocyanins from Muscadine [23]. 3.3. Adsorption equilibrium and kinetics

Fig. 1. Adsorption and desorption capacities of jamun anthocyanins on different adsorbents.

Adsorption is known to be a physio-chemical process that involves the mass transfer of a solute (adsorbate) from the fluid

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Fig. 5. (a) First order kinetic model (b) Second order kinetic model. qt – adsorption capacity at time ’t’, qe – adsorption capacity at equilibrium.

Fig. 4. (a) Langmuir plot and (b) Freundlich plot for adsorption of anthocyanins. qe – adsorption capacity (mg/mL of adsorbent) at equilibrium, Ce – concentration of anthocyanins (mg/mL) at equilibrium.

25 15 5

Langmuir constants

Model

Pseudo first order Pseudo second order

Table 2 Langmuir and Freundlich constants at different temperatures. Temperature (°C)

Table 3 Adsorption kinetic model rate constants.

Freundlich constants

KL

qmax

R2

KF

n

R2

0.005 0.009 0.015

1.25 0.9 0.89

0.999 0.997 0.988

1.19 1.09 1.05

5.71 5.31 4.04

0.967 0.896 0.875

qmax – maximum adsorption capacity. R2 – regression coefficient. KL – Langmuir constant. KF – Freundlich constant. n – adsorption intensity.

phase to the adsorbent surface. A study of kinetics of adsorption is desirable as it provides information about the mechanism of adsorption, which is important for evaluating the efficiency of the process. The study of adsorption kinetics describes the solute uptake rate at the solid-solution interface. 3.3.1. First order kinetic model In order to evaluate the applicability of pseudo-first order kinetic model for the adsorption of anthocyanins, a linear plot of log(qe–qt) vs t is obtained as shown in Fig. 5a. The values of qe (obtained from the model) and k1 obtained from the intercept and slope of the plot, respectively are presented in Table 3. It can be seen from the table that the experimental adsorption capacity (1.07 mg/mL) is almost equal to qe (1.10 mg/mL) obtained from

Rate constants k1 (or) k2

qe

R2

k1 = 0.08 k2 = 0.038

1.1 1.3

0.937 0.995

k1 – first order rate constant. k2 – second order rate constant. qe – adsorption capacity of the kinetic model. R2 – regression coefficient.

this model. However, the value of R2 (0.937) is much lower than unity, from which it can be inferred that the adsorption of jamun anthocyanins does not follow the first order kinetics. 3.3.2. Second order kinetic model The linear plot of t/qt versus t is shown in Fig. 5b. The equilibrium adsorption capacity and the second order constant (k2) determined from the slope and intercept of the plot, respectively are presented in Table 3. It can be seen from the table that the R2 (0.995) is close to unity and qe (1.3) obtained from this model is close to the experimental adsorption capacity (1.07). Hence, it can be inferred that second order kinetic model is more appropriate to describe the adsorption of anthocyanins. 3.4. Adsorption mechanism Identification of the rate limiting step is an important factor to be considered in adsorption process. The solute transfer was usually characterized by either external mass transfer (boundary layer diffusion) or intraparticle diffusion or both. The overall sorption

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Fig. 6. Intra-particle diffusion model. qt – adsorption capacity at time ’t’.

Fig. 7. Effect of alcohol concentration (%, v/v) on the elution of anthocyanins.

rate is shown to be controlled by several factors including the following steps [34]. (i) Diffusion of the solute from the solution to the film surrounding the particle. (ii) Diffusion across the film to the particle surface (external diffusion). (iii) Diffusion from the surface to the internal sites (pore diffusion). Adsorption can involve several mechanisms such as physicochemical sorption, ion exchange and precipitation or complexation [35,36]. Intra-particle diffusion equation usually employed to model sorption processes [37,38] is given as

qt ¼ K p t1=2 þ C

ð16Þ 1

where qt is the amount of anthocyanins adsorbed (mg g ) at time t; C is the boundary layer thickness and Kp is the intra-particle diffusion rate constant (mg g1 min1/2). The plot of qt versus t1/2 is shown in Fig. 6. It can be observed from the figure that R2 value is high (0.986) and the plot of qt vs. t0.5 giving straight line but not going through the origin. It indicates that although intra-particle diffusion was involved in the adsorption process, it was not the sole rate controlling step. Similar results were reported in case of adsorption of anthocyanins from Hibiscus sabdariffe L. [24]. 3.5. Elution of anthocyanins: Effect of ethanol concentration In order to know the effect of ethanol concentration on the elution of anthocyanins, studies were carried out with acidified

Fig. 8. (a) Dynamic break-through and (b) dynamic desorption of anthocyanins.

Table 4 Evaluation of stability of anthocyanins. Components

Crude extract

After purification

pH Soluble solids (°B) Sugars (lg/mL) Non-enzymatic browning (–) Degradation constant (day1) L a b Hue Chroma

2.8 4 175.32 0.45 0.93 23.43 31.23 21.67 34.76 38.01

2.1 12 0.12 0.29 0.19 25.35 41.02 28.35 34.65 49.86

aqueous ethanol (with 1% v/v HCl and ethanol concentration varying from 20–100% v/v) and the results are presented in Fig. 7. It can be seen from the table that acidified ethanol of concentrations above 40% (v/v) could effectively elute anthocyanins from the adsorbent. Hence, higher concentration (>40% v/v) of ethanol was used for further experiments. 3.6. Dynamic adsorption and desorption curves on Amberlite XAD7HP Process parameters such as volume of feed and eluent, flow rate of feed and eluent were taken into consideration to standardize the dynamic adsorption and desorption of jamun anthocyanins. In order to standardize the volume of feed required and the flow rate, experiments were carried out to calculate the break-through point (the point beyond which the concentration of anthocyanins in

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C. Jampani et al. / Separation and Purification Technology 125 (2014) 170–178 2: Diode Array 530 Range: 4.404e-2

4.0e-2

AU

3.23

(a)

2.0e-2

7.27

5.70

3.07

9.50

11.55 18.52

0.0 0.00

90

2.50

5.00

7.50

10.00

15.00

17.50

20.00

22.50 25.00 1: TOF MS ES+ TIC 2.51e3

15.00

17.50

20.00

22.50

%

1.73

7.39 8.03 9.97

-10 0.00

2.50

5.00

7.50

10.66 12.23

10.00

(6.355)

100

10.31

12.50

Time 25.00

1: TOF MS ES+ 314

627.3439

%

(b)

12.50

0 100

300

400

500

700

800

900

1000

1100

1200

1300

641.3630

(8.028)

100

600

1400

1: TOF MS ES+ 2.35e3

%

(c)

m/z 200

427.2682

0 100

300

400

(8.617)

100

500

600

700

800

900

1000

1100

1200

1300

1400 1: TOF MS ES+ 365

465.3258

%

(d)

611.3318

m/z 200

460.3553

0 100

100

300

400

x5

500

600

700

800

900

1000

1100

1200

1300

(10.307) 655.3804

1400 1: TOF MS ES+ 7.10e3

305.1615

%

(e)

m/z 200

656.3928

489.2623

0 100

300

400

(12.232)

100

500

600

700

800

900

1000

1100

1200

1300

1400 1: TOF MS ES+ 925

465.2467

%

(f)

m/z 200

0 100

m/z 200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

Fig. 9. (a) HPLC Chromatogram of anthocyanins and (b–f) corresponding molecular mass of peaks.

fractions increases) at different flow rates of feed (1, 2 and 3 mL/ min). The dynamic breakthrough curve on Amberlite XAD7HP resin was obtained based on the volume of eluent liquid and the concentration of solute therein and is given in Fig. 8a. The best adsorption performance was observed at the lowest flow rate 1 mL/min. Low flow rate allows more time for the adsorbate molecules to interact with the active sites of the adsorbent. In contrast, high flow rate re-

quires less time but has a negative impact on the adsorption capacity since the residence time of anthocyanins is less. Hence, the break-through point is reached more quickly. In general, the eluent concentration on reaching the 5% of the inlet concentration is defined as the breakthrough point [23]. Hence, it can be inferred that 6.5 BV of solution and flow rate of 1 mL/min are the standardized conditions for dynamic adsorption.

C. Jampani et al. / Separation and Purification Technology 125 (2014) 170–178 Table 5 Chromatographic and spectroscopic characteristics, and composition of anthocyanins from jamun, obtained by HPLC–DAD–MS/MS. Figure No.

tr (min)

kmax (nm)

[M]+ (m/z)

Anthocyanin

9b 9c 9c 9d 9e 9f

6.35 8.02 8.02 8.61 10.3 12.23

275, 528 276, 528

627 611 641 465 655 465

Delphinidin 3,5-diglucoside Cyanidin 3,5-diglucoside, Petunidin 3,5-diglucoside Delphinidin 3-glucoside Malvidin 3,5-diglucoside Delphinidin 3-glucoside

274, 517 275, 528 275, 532

tr – retention time.

The dynamic desorption curve was obtained based on the volume of desorption solution and the concentration of solute therein and is given in Fig. 8b. Acidified aqueous ethanol (40%, v/v) was used for elution of anthocyanins by maintaining the flow rates at 1 and 2 mL/min. It can be seen from the figure that approximately 4 BV desorption solution could completely elute the anthocyanins at a flow rate of 1 mL/min whereas 6 BV desorption solution at a flow rate of 2 mL/min. Hence, 4 BV desorption solution and 1 mL/ min flow rate are inferred as the most suitable conditions for the desorption of jamun anthocyanins. The optimum adsorption process parameters for the purification of anthocyanins with Amberlite XAD7HP arrived at are as follows; for adsorption: processing volume, flow rate and temperature are 6.5 BV, 1 mL/min and 25 ± 1 °C, respectively and for desorption: eluent volume and flow rate are 4 BV of acidified aqueous ethanol (40%, v/v) and 1 mL/min, respectively. 3.7. Evaluation of anthocyanins solution Besides anthocyanins, jamun extract contains other components also. Hence, in order to examine the final product (anthocyanins) stability, evaluation was carried out with respect to total sugars, soluble solids and pH of the anthocyanins extract before and after adsorption with Amberlite XAD7H under saturated adsorption conditions. In addition, the colorant properties browning index, degradation constant and Hunter L, a, b, values were evaluated. The results are presented in Table 4. It can be observed from the table that the concentration of soluble solids increased from 4° to 12° B after purification. The concentration of sugars (the major cause for product degradation) was observed to decrease from an initial 175.32 to 0.12 lg/mL after purification. The degradation constant of anthocyanins was observed to decrease from 0.93 to 0.19 signifying the increased stability of anthocyanins. Non-enzymatic browning index was also observed to decrease from 0.45 to 0.29. 3.8. HPLC–MS/MS analysis HPLC–MS/MS was carried out in order to know the basic structure as well as structural stability of anthocyanins and the results are presented in the Fig. 9. It can be observed from the figure that the molecular mass of the peaks obtained during the mass spectrum are equal to the reported values [33,39]. It can be inferred that the structure of pigments did not get affected during the process. The chromatographic and spectroscopic characteristics, and composition of anthocyanins from jamun obtained by HPLC– DAD–MS/MS are given in Table 5. It can be observed from the table that five different anthocyanins could be identified in the jamun. 4. Conclusions Purification of jamun anthocyanins (removal of free sugars) could be done successfully employing adsorption. Amberlite XAD7HP showed highest adsorption capacity (1.07 mg/mL of

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adsorbent) and desorption capacity (87.62%), among the all adsorbents studied. Adsorption of anthocyanins correlated best using Langmuir isotherm. Second order kinetic model was found to be most appropriate to explain the adsorption of anthocyanins onto the Amberlite XAD7HP. Dynamic adsorption and desorption process parameters were successfully standardized. Evaluation of the purified anthocyanins with respect to total sugars, total soluble solids, degradation constant and non-enzymatic browning index ensured the increased stability of purified anthocyanins. HPLC– MS/MS results confirmed the structural stability of purified anthocyanins. Acknowledgment Authors thank the Director, CFTRI, Mysore, for the infrastructural facilities at the institute. Chandrasekhar Jampani and Aduja Naik gratefully acknowledge CSIR and UGC, Government of India, respectively for their research fellowship. References [1] Pu Jinga, Si-Yu Ruan, Ying Dong, Xiao-Guang Zhang, Jin Yuea, Jian-Quan Kanc, Margaret Slavina, Liangli Yua, Optimization of purification conditions of radish (Raphanus sativus L.) anthocyanin-rich extracts using chitosan, LWT – Food Science and Technology 44 (2011) 2097–2103. [2] G.G. Cristina, Emilie Destandau, Sandrine Zubrzycki, Claire Elfakir, Sweet cherries anthocyanins: an environmental friendly extraction and purification method, Sep. Purif. Technol. 100 (2012) 51–58. [3] G.J. Mazza, Anthocyanins and heart health, Ann. Ist. Super. Sanita. 43 (2007) 369–374. [4] B. Jayaprakasam, L.K. Olson, R.E. Schutzki, M.H. Tai, M.G. Nair, Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas), J. Agric. Food Chem. 54 (2006) 243–248. [5] C. Andres-Lacueva, B. Shukitt-Hale, R.L. Galli, O. Jauregui, R.M. LamuelaRaventos, Anthocyanins in aged blueberry-fed rats are found centrally and may enhance memory, Nutr. Neurosci. 8 (2005) 111–120. [6] L.S. Wang, G.D. Stoner, Anthocyanins and their role in cancer prevention, Cancer Lett. 269 (2008) 281–290. [7] M.S.M. Rufino, R.E. Alves, E.S. de Brito, J. Pe´rez-Jime´nez, F. Saura-Calixto, J. Mancini-Filho, Bioactive compounds and antioxidant capacities of 18 nontraditional tropical fruits from Brazil, Food Chem. 121 (2010) 996–1002. [8] M.S.M. Rufino, R.E. Alves, F.A.N. Fernandes, E.S. Brito, Free radical scavenging behavior of ten exotic tropical fruits extracts, Food Res. Int. 44 (2011) 2072– 2075. [9] Manjeshwar Shrinath Baliga, Harshith P. Bhat, Bantwal Raghavendra Vittaldas Baliga, Rajesh Wilson, Princy Louis Palatty, Phytochemistry, traditional uses and pharmacology of Eugenia jambolana Lam. (black plum): a review, Food Res. Int. 44 (2011) 1776–1789. [10] P. K. Warrier, V.P.K. Nambiar, C. Ramankutty, Indian Medicinal Plants, vol. 5, India: Orient Longman Ltd., Hyderabad, 1996, pp. 225–228. [11] K.M. Nadkarni, in: Indian Materia Medica, Dhootapapeshwar Prakashan Ltd., India, 1954, p. 516. vol. 1. [12] J. Morton, Fruits of Warm Climates, Miami, 1987, p. 375. [13] S. Achrekar, G.S. Kakliji, M.S. Pote, S.M. Kelkar, Hypoglycemic activity of Eugenia jambolana and Ficus bengalensis: mechanism of action, In vivo 5 (2) (1991) 143–147. [14] Farrukh Aqil, Akash Gupta, Radha. Munagala, Jeyaprakash. Jeyabalan, Hina. Kausar, Ram Jee Sharma, Inder Pal Singh, Ramesh C. Gupta, Antioxidant and antiproliferative activities of Anthocyanin/Ellagitannin-enriched extracts from syzygium cumini L. (Jamun, the Indian Blackberry), Nutr. Cancer (2012) 1–11. [15] K.F. Migliato, Standardization of the extract of. Syzygium cumini (l.) skeels fruits. through the antimicrobial activity, Caderno de Farma´ cia 21 (1) (2005) 55–56. [16] L. Li, L.S. Adams, S. Chen, C. Killian, A. Ahmed, N.P. Seeram, Eugenia jambolana Lam. berry extract inhibits growth and induces apoptosis of human breast cancer but not non-tumorigenic breast cells, J. Agri. Food Chem. 57 (2009) 826–831. [17] F.J. Francis, Food colorants: anthocyanins, Crit. Rev. Food Sci. Nutr. 28 (1989) 273–314. [18] J.B. Wan, Q.W. Zhang, W.C. Ye, Y.T. Wang, Quantification and separation of protopanaxatriol and protopanaxadiol type saponins from Panax notoginseng with macroporous resins, Sep. Purif. Technol. 60 (2) (2008) 198–205. [19] Chaoyang Ma, Guangjun Tao, Jian Tang, Zaixiang Lou, Hongxin Wang, Xiaohong Gu, Liming Hu, Menglong Yin, Preparative separation and purification of rosavin in Rhodiola rosea by macroporous adsorption resins, Sep. Purif. Technol. 69 (1) (2009) 22–28. [20] X. Liu, G. Xiao, W. Chen, Y. Xu, J. Wu, Quantification and purification of mulberry anthocyanins with macroporous resins, J. Biomed. Biotechnol. 5 (2004) 326–331.

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