Ultrasonic assisted aqueous extraction of catechin and gallic acid from Syzygium cumini seed kernel and evaluation of total phenolic, flavonoid contents and antioxidant activity

Journal Pre-proof Ultrasonic assisted aqueous extraction of catechin and gallic acid from Syzygium cumini seed kernel and evaluation of total phenolic, flavonoid contents and antioxidant activity Komal V. Mahindrakar, Virendra K. Rathod

PII:

S0255-2701(19)30701-9

DOI:

https://doi.org/10.1016/j.cep.2020.107841

Reference:

CEP 107841

To appear in:

Chemical Engineering and Processing - Process Intensification

Received Date:

12 June 2019

Revised Date:

23 December 2019

Accepted Date:

27 January 2020

Please cite this article as: Mahindrakar KV, Rathod VK, Ultrasonic assisted aqueous extraction of catechin and gallic acid from Syzygium cumini seed kernel and evaluation of total phenolic, flavonoid contents and antioxidant activity, Chemical Engineering and Processing - Process Intensification (2020), doi: https://doi.org/10.1016/j.cep.2020.107841

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Ultrasonic assisted aqueous extraction of catechin and gallic acid from Syzygium cumini seed kernel and evaluation of total phenolic, flavonoid contents and antioxidant activity

Komal V. Mahindrakar, Virendra K. Rathod*

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*Corresponding Author

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Dr. Virendra K. Rathod Department of Chemical Engineering,

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Institute of Chemical Technology, Matunga (E), Mumbai-400019, India.

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E-mail: [email protected],

Phone: +91-22-33612020, Fax: 91-22-33611020.

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Highlights

Catechin and gallic acid extraction from S. cumini were intensified by ultrasound.

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The effect of important extraction parameters were checked to increase efficiency.

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TPC, TFC and antioxidant assays of extract were carried out.

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Antioxidant phenolics were stable under mild ultrasonic conditions.

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Extraction by cavitation was superior over conventional techniques.

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Abstract Present work reported ultrasonic-assisted aqueous extraction (UAE) of bioactives from Syzygium cumini seed kernels powder (SCSKP) as an alternative to conventional, soxhlet and stirred batch techniques. The yield of catechin, gallic acid, TPC (Total phenolic contents), TFC (Total flavonoid contents) and IC50 value obtained was 2.2, 54.5 mg/g, 100.07 mg of gallic acid equivalent (GAE)/g of SCSKP, 10.11 mg of catechin equivalent (CE)/g of SCSKP and 10.59

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µg/mL respectively in 12 min. extraction time when other optimum parameters were used such

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as solute to the solvent ratio 1:15, temperature 35°C, power 125W, and duty cycle 60% (1.2 min ON and 0.8 min. OFF). TPC, TFC and antioxidant activity of the extract were determined by

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using Folin Ciocalteu (FC), AlCl3 and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. The

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optimized stirred batch technique was used for sequential batch extraction till 5 stages giving catechin and gallic acid yield of 1.05 mg/g and 53.3 mg/g, respectively. UAE technique not only

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increased the yield of catechin, gallic acid, TPC, TFC by 3.6, 1.5, 1.3, 1.4 times respectively and antioxidant activity by 1.2 times but also reduced the extraction time, amount of solvent and

technique.

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temperature by 8.8, 1.3 and1.4 times respectively as compared to conventional stirred batch

Keywords: Catechin, Gallic acid, Syzygium cumini, Sequential batch extraction, ultrasound

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assisted extraction, phenolic antioxidants.

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1. Introduction The waste parts of many plants are rich in polyphenols having high antioxidant activity. The use of natural antioxidants (derived from plants and herbs) is seeking attention recently due to probable queasy properties of synthetic antioxidants available in the market. Polyphenols are herbal antioxidant compounds, along with multiple medicinal effects [1] [2] [3]. Syzygium

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cumini Linn. is a widespread, attractive tree species belongs to the subdivision spermetophytina and family Myrtaceae. The tree is known scientifically as Eugenia cuminii Druce or Eugenia

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jambolana Lam or Syzygium jambolana Dc. The homeplace of S. cumini tree is India and its

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local names are Java Plum, Jambhul, Indian Blackberry, Jamun, Rose apple, Black Plum, Jamblang, etc. Extract of all parts of S. cumini tree possesses tremendous pharmacological

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properties. The therapeutic properties of the herbal extract are dependent on their constituents. Phytoconstituents present in seed are reported as gallic acid, 1-galloyl glucose, corilagin, 3,6-

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hexahydroxy diphenoyl glucose, 3-galloyl glucose, quercetin, β-sitosterol, ellagic acid, 4,6 hexahydroxy diphenoyl glucose [4] 1−3, oenothein C, cornussin B, and swertisin, valoneic acid

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dilactone, brevifolin [5]. Seeds possess antioxidant [6], antidiabetic, antiulcer [7], antibacterial [8] properties.

Catechin belongs to family flavonoids, ((2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1

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(2H)-benzopyran-3,5,7-triol) is a flavan-3-ol, medicinally active polyphenolic compound [9]. Gallic acid is a naturally occurring simple polyphenol, chemically 3, 4, 5-trihydroxy benzoic acid. Catechin and gallic acid are widely used in neutraceuticals. Some major sources of catechin and gallic acid are grapes [10], tea [11], apple [12], pear [13], apricots [14], strawberry, blackberry and raspberry leaves [15]. Catechin and gallic acid show antidiabetic [16] [17],

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antioxidant [18] [19], anticancer [20] [21], antimicrobial [22] [23], anti-inflammatory [24] [25], antimutagenic [26] [27] properties. In solid-liquid extraction, solvent extraction of solid samples is carried out to get valueadded products. The general techniques of extraction of constituents from the natural source include heat flux solvent extraction (Soxhlet), infusion, maceration but, they encounter various problems. Green extraction techniques are becoming most attractive due to the evolution of

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“Green chemistry’ principles. For the last few years, other investigations with new techniques

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such as microwave-assisted [28], ultrasound assisted extraction (sonication) [29], and extraction by supercritical carbon dioxide [30], subcritical extraction [31] are also emerging to isolate

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important herbal components. The application of ultrasonic waves for the extraction of high-cost

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medicinally important phytoconstituents is a better alternative. Definite ultrasonic conditions can also extract securely thermolabile and unstable phytoconstituents. Intensification of an extraction

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efficiency of phytocompounds by sonication is mainly due to the cavitation, mechanical and thermal effects that occur in the solvent by the transit of ultrasonic waves which can enhance the

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mass transfer by disrupting cell walls and reducing particle size [32]. Kernels are generated abundantly from Syzygium cumini fruit processing industries which are less expensive and can be collected easily. Suitable valorization of the food industry byproduct avoids the disposal as a waste because it causes serious economic and environmental

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problems such as groundwater contamination [33] [34] [35]. The restraint of these residues would ameliorate the technique both economically and environmentally, making it green. The concentration of phytocompounds present in crude is based on the type of soil, season and crop load, etc. [36]. The yield of phytochemicals by extraction is mainly dependent on operating conditions under which the process is conducted and solvent type [37]. This study aims to gain

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insight into the role of factors potentially affecting the UAE of catechin and gallic acid, and further evaluation of total phenolic contents (TPC), total flavonoid contents (TFC) and antioxidant activity. The sequential batch extraction procedure was performed by keeping previously optimized experimental conditions to get an improved yield of catechin and gallic acid by repeatedly using the same residue material obtained from the first stirred batch extraction. In this study, the pH of the mixture wasn’t adjusted because phytocompounds are

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published yet in the past to the best of our knowledge and information.

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mostly stable at acidic conditions only [38] [39]. This kind of study has not been conducted and

2. Materials and Methods

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2.1. Materials

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Syzygium cumini seed kernels were obtained from the fruit processing industry located at Konkan region of Maharashtra, India. The kernels were washed, sun-dried, ground and stored in

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an airtight container in a cool place. The S. cumini seeds kernel powder (SCSKP) is brown having a characteristic odor with moisture content NMT 5% and the particle size range of about

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106 µm. Catechin (≥99.05%), Aluminium chloride, FC reagent, and sodium carbonate were procured from Total Herb Solutions Pvt Ltd., Thomas Baker, Fisher Scientific, and Merck respectively. Gallic acid (≥ 98% HPLC grade) and DPPH were procured from Sigma Aldrich. Millipore Milli-Q 50 HPLC grade water was used for all experimental studies. All used solvents

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were of AR grade and purchased from Hi-Media Ltd. (Mumbai, India). 2.2. Experimental work 2.2.1 Soxhlet extraction Soxhlet extraction was conducted according to the method described previously with little modifications [29]. In brief, 1 g SCSKP was wrapped in filter paper and put in a soxhlet

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thimble holder. Water was loaded in a round bottom flask and the soxhlet apparatus was placed in a heating mantle as a heating source. The extraction was performed for 6 h and the extract was analyzed by HPLC technique. The yield of catechin and gallic acid was calculated in mg/g. 2.2.2 Batch extraction Batch extraction was conducted in a 50 ml glass reactor furnished with three bladed (pitched blade) glass turbines for stirring. The required amount of the SCSKP was kept in a glass

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reactor, and the measured amount of solvent (water) was poured to it. The extraction mixture

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was then stirred in the reactor for 105 min. with 250 rpm impeller speed, solute to the solvent ratio 1:20, temperature 50˚C, particle size 106 µm. Centrifugation of the extraction mixture was

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carried out to get separate the extract and marc. The extract was filtered by using a nylon syringe

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filter having 0.45 µm pore size. The clear extract was then diluted 10 times and analyzed for catechin and gallic acid using HPLC technique and calculated the yield in mg/g.

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2.2.3 Ultrasound assisted aqueous extraction

UAE was carried out by using ultrasound bath (model no. 6.5l 250H/DTC/DF) having 22

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and 40 kHz operating frequencies and power 215 W. Extraction experiments were carried out by adding a measured quantity of SCSKP and water as a solvent in a cylindrical flat bottom glass vessel of known dimensions. Vessel kept in an ultrasound bath at the known position which had been optimized earlier [40]. The mixture was then sonicated for 12 min. with the solute to the

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solvent ratio 1:15, frequency 22 kHz, temperature 35°C, duty cycle 60%, and power 125 W. Sampling was done regularly after a certain period of time and centrifuged. The extract was filtered by using a nylon syringe filter having 0.45 µm pore size. The clear extract was diluted 10 times and catechin and gallic acid analyzed by using HPLC analytical technique to determine the yield. Different important parameters affecting the UAE, such as time for extraction (0-20

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minutes), solute to solvent ratio (1:05 to 1:50), temperature (25-65 ˚C), duty cycle (10-100%), power (44-215 W), were optimized to get the maximum recovery of the catechin and gallic acid. 2.2.4 Total Phenolic Content An assessment of total phenolics in the soxhlet, batch, and ultrasonic extract was studied by using Folin–Ciocalteu reagent. In brief, 100 µL of the test sample and 100 µL of Folin– Ciocalteu reagent were mixed properly. After 4 min., 2 mL of 7.5% Na2CO3 was added, and then

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the obtained mixture was incubated for 2 h. The absorbance of all samples was measured at 765

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nm using Jasco V-630 spectrophotometer. Various concentrations (10-1000 mg/L) of standard gallic acid were prepared in water. Then the same assay protocol was repeated and plotted a

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calibration curve. TPC of the extract was evaluated on the basis of gallic acid equivalent (mg of

formula: GAE × V × D W

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Total phenolic content =

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GAE/g of SCSKP). Total phenolic content of SCSKP extract was calculated using the following

(1)

Where, GAE - Gallic acid equivalent (mg/L) obtained by using the slope and constant from

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calibration curve; D - Dilution Factor; V - Volume of solvent used for extraction (mL); W SKSKP weight (g).

2.2.5 Total flavonoid content

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The AlCl3 method for flavonoid content estimation in the soxhlet, batch, and ultrasonic extract employed was based on the protocol described by Stankovic [37]. Aqueous extract of 200 µL was added to 200 µL of 2% methanolic AlCl3. The mixture was diluted with 600 µL water. After agitation, the absorbance was measured 1 h later at 415 nm by using Jasco V-630 spectrophotometer. Catechin was used as a reference flavonoid. Various concentrations (10-100 mg/L) of catechin were prepared in methanol. Then the same assay procedure was repeated and 7

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plotted a calibration curve. Total flavonoid content of SCSKP extract was determined using the following formula: Total flavonoid content =

CE × V × D W

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Where CE - catechin equivalent (mg/L) obtained by using the slope and constant from calibration curve; V - Volume of solvent used for extraction (mL); D - Dilution Factor; W -

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SCSKP weight (g).Total flavonoid contents of extract were evaluated in terms of catechin equivalents (mg of CE/g of SCSKP).

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2.2.6 In vitro antioxidant activity

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The extracts obtained by soxhlet, batch, and ultrasound-assisted extraction technique were taken to perform the antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl radical-

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scavenging (DPPH) assay as per the protocol followed by Kulkarni and Rathod with slight

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modifications [41]. In brief, 100 µL of extract and 1.5 ml of DPPH (1.6 mg DPPH in 50 ml of 80% ethanol) solution were mixed thoroughly then the mixture was incubated for 1 h in the dark. The absorbance of 80% ethanol was considered as the blank. The percentage of antioxidant

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activity at different concentrations (10-100 mg/L) of the SCSKP extract was determined. Catechin and gallic acid were used as the reference standard. The % free radical scavenging activity was calculated by using the given equation:

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% Antioxidant activity =

Acontrol − Aextract × 100 Acontrol

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Where, A control - Absorbance of control; A extract - Absorbance of extract. The percentage of antioxidant activity versus the concentration of the extract was plotted. IC50 value (µg/mL) was got by interpolation from the logarithmic regression analysis.

2.3 Analysis of catechin and gallic acid

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Analysis of catechin and gallic acid was done by Jasco HPLC system fitted with InertClone column (5 µ x 4.6 mm x 250 mm). The column for HPLC was equilibrated with methanol-acetonitrile-water (10:10:80) mixture as a mobile phase by isocratic elution at 1 mL/min flow rate. Catechin and gallic acid were detected by measuring UV absorption at 210 nm. The amount of catechin and gallic acid present in the extract were quantified by using slope obtained from the respective standard curves. All the experiments were carried out thrice to

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study the reproducibility, and their average values are reported. One-way ANOVA was used for

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statistical analysis and got the p values. The values were considered statistically significant if the p values were <0.05.

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3. Results and discussion

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3.1 Effect of time

The time is a key point for minimization of cost and development of a better process.

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Effect of time parameter on the UAE of catechin and gallic acid from SCSKP was studied using water as a green solvent. The other process parameters were kept constant, i.e. solid to solvent

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ratio 1:20, temperature 25 ºC, frequency 22 kHz, and ultrasound power of 100 W. Results depicted in Fig. 1 show the amount of catechin and gallic acid extracted per gram of SCSKP with time. The rate of extraction was initially higher, and gradually increased up to 8 min. for catechin and 12 min. for gallic acid and remained constant for an extended time. The initial higher

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extraction rate was due to the higher concentration difference, which further reduced with time. In our study, when the water exposed to ultrasonic waves, developed the emergence of vicious bubbles that could generate the utmost mechanical shear by collapsing bubbles. This made possible the cell membrane disruption and release of catechin and gallic acid. Rapid extraction

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was achieved due to the cavitational, physical and thermal effects generated at cell walls [42]. Hence, 12 min. was selected as the optimum time for the extraction of catechin and gallic acid. 3.2 Effect of SCSKP to water ratio The role of solid to solvent ratio has a significant influence on extraction efficiency. Hence, the effect of SCSKP to water ratio was varied from 1:05 to 1:50 and the results are shown in Fig. 2. It was noticed that the yield of catechin and gallic acid extracted per gram of SCSKP

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increases from 1:05 to 1:15 ratio. Sufficient solvent volume is required to enable proper

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hydration and swelling of solute. This will help in the solvation of the phytoconstituents from the extracting material more efficiently as a higher concentration gradient favors the mass transfer.

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The almost similar yield of catechin and gallic acid was achieved by using 1:15 and 1:20 solute

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to solvent ratios. Afterward, an insignificant change in the yield was observed. Solute loading in the extraction process decides the transfer of energy on the cell’s surface. It was observed from

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the literature that the use of more amount of SCSKP could increase the surface tension and viscosity, which causes difficulty in cavitation [43]. Hence, to avoid the excessive use of water

studies.

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and bulky handling of the process, 1:15 ratio was decided as optimum and used for remaining

3.3 Effect of temperature

Conventional extraction techniques are usually performed at higher temperatures.

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Temperature affects extraction significantly because it controls the solubility, mass transfer rate of target compounds in solvent and cavitation. The effect of temperature on ultrasound-assisted extraction was studied. In this experiment, six different temperatures (25, 30, 35, 45, 55 and 65ºC) were chosen to study the influence of temperature on the extraction yield of catechin and gallic acid from SCSKP. Other conditions were solute to the solvent ratio, 1:15, ultrasound

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power 100 W, duty cycle 50 % and extraction time 12 min. The highest extraction yields of catechin and gallic acid were obtained at 35 ºC (Fig. 3). High temperature increases the solubility and mass transfer across the cell, which increases the extraction yield. However, a further increase in temperature for extraction from 35 ºC to 65 ºC doesn’t show a significant increase in the yield. In the UAE, up to a certain limit, the cavitational and thermal effects are responsible for the increase in extraction yield. Further increase in temperature the cavitation intensity

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decreases, which could be due to increased vapor pressure and reduced surface tension. The rise

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in temperature leads to a lowering the viscosity of the solvent, which causes increased vapor pressure resulting in the formation of more bubbles. However, due to the lesser pressure

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difference among the inner and outer side of the bubbles, they collapse with lesser intensity. This

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is the logical reason for ultrasound being less prominent at higher temperatures, and any advantage should not just be referred to its effect [42]. Hence, 35 ºC was used as the optimized

3.4 Effect of power

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extraction temperature for the remaining set of ultrasonic experiments.

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Ultrasonic power is a crucial operational parameter in process optimization of UAE, because ultrasonic vibration is proportional to its power for given media and a fixed radiation area. Higher power gives more cavitation in the medium due to larger amplitude ultrasonic waves generation. The pressure and temperature are very elevated inside the bubble generated

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during cavitation. When the bubble burst, the violent shock wave along with the high-speed jet was produced, which facilitates cell wall disruption and intensifies the penetration of the water into the cell and delivers the intracellular compounds into it. Fig. 4 shows that the extraction yield of catechin and gallic acid increased by 3.7 and 2.1 times respectively with an increase in the ultrasonic power from 44 W to 125 W. However, the increase in yield of both the compounds

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was not very significant over ultrasound power 125 to 215. Since the ultrasound power increases, there is a large amplitude of wave traveling through the liquid media, the greater number of bubbles are formed and crumbled more violently with enhanced cell disruption with the release of intracellular components [43]. To determine the real power consumption during extraction, the power dissipation has been determined by the calorimetric study. The power dissipation at 44, 79, 102, 125, 168, 215 W were 20.65, 29.05, 32.9, 38.15, 50.75, 62.3 W respectively. Hence, to

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for further extraction experimentation of catechin and gallic acid.

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avoid excessive electricity consumption, 125 W ultrasound power was considered as optimum

3.5 Effect of the duty cycle

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Ultrasonic irradiation causes the enhanced mass transfer by decreasing resistances across

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the cell and solvent, which facilitates the release of intracellular material to the exterior environment. Continuous mode of ultrasonic irradiation causes higher temperature generation,

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which enhances the cost of the process and may degrade the phytoconstituents due to continuous exposure of the cell to cavitation. Duty cycle is the cycle of operation of ultrasound which

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operates in pulse mode (‘ON’ and ‘OFF’) [44]. To get the optimum value, the duty cycle for sonication was studied by setting different ON-OFF times when the total time of one cycle was fixed to 2 min. Other operational parameters were kept at constant values such as temperature 35ºC, the SCSKP to solvent ratio 1:15, time 12 min., power 125 W, at 22 kHz frequency. The

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duty cycle has a prominent impact on the extraction of catechin and gallic acid, as can be seen in Fig. 5. It shows that the extraction yield of catechin and gallic acid was increased by almost 22 and 18 times, respectively with the duty cycle from 10 to 60 %. There was no further rise in the extraction yield when experiments were carried out up to 100% duty cycle. Hence, for higher

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extraction yield, 60% duty cycle (1.2 min. ‘ON’ and 0.8 min. ‘OFF’) was considered to be optimum. 3.6 Total phenolic and total flavonoid contents Presence of certain phenolic compounds having one or more aromatic rings along with one or more hydroxyl groups in their structure such as gallic, ellagic, caffeic, p-coumaric acid, catechin, epicatechin, quercetin, tannic acid in S. cumini seed extract is reported by Balyan and

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Sarkar [45] [46]. The effect of extraction time on TPC and TFC by keeping other previously

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optimized parameters for catechin and gallic acid extraction constant such as power 125 W, temperature 35ºC, the solute to solvent ratio 1:15, at 22 kHz frequency up to 16 min. were

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studied. TPC and TFC of the aqueous ultrasonic extract were measured by Folin Ciocalteu and

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AlCl3 assays, respectively. As time increased from 0 to 12 min., TPC and TFC were also increased readily, i.e., 100.07 mg of GA/g of SCSKP and 10.11 mg of CE/ g of SCSKP

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respectively and marginal change in yield was observed after 12 min. of extraction time (Fig. 6). The same time duration was also required to get the optimum yield of catechin and gallic acid

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(Fig. 1). It proves that the phenolic compounds were extracted majorly in this time span. 3.7 Antioxidant activity

The more amount of phenolics relates to higher antioxidant activity [47]. Antioxidants are secondary metabolites and their activity relies on the structure of any compound and position

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of hydroxyl groups [48]. Antioxidant intake prevents many health issues by controlling oxidative stress like lower immunity [49]. Recently, herbal antioxidants have achieved greater attention than allopathic [50]. Free radical scavenging activity of SCSKP extract obtained by soxhlet, batch and UAE was determined by the reduction in color of ethanolic DPPH solution. Serial dilutions of standard catechin, gallic acid, ultrasonic aqueous extract were prepared in 10, 25, 50,

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75 and 100 ppm of the concentration. The IC50 values of the soxhlet, batch, ultrasonic aqueous extract, standard gallic acid, and standard catechin were 35.89, 12.97, 10.59, 5.9, 5.35 µg/mL respectively. IC50 is the concentration of the extract at which half the DPPH radicals will be scavenged. A lower IC50 value corresponds to a greater antioxidant potential. The ultrasonic aqueous extract is showing less IC50 value compared to conventional techniques suggesting more

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extraction of antioxidants [51]. Fig. 7 shows a comparative plot of concentration of extract vs % antioxidant activity of catechin, gallic acid, and ultrasonic aqueous extract. Antioxidant capacity

extracts play an important role in antioxidant properties.

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of extract and standards are comparable, which confirms that catechin and gallic acid in the

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4. Comparison of ultrasonic aqueous extraction and conventional extraction techniques

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Comparisons were made between advance ultrasonic and conventional, soxhlet and stirred batch extraction techniques reflected by yields of total phenolics, flavonoids, catechin,

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gallic acid and IC50 values of the extract obtained by subsequent techniques and its operating conditions (Table 1 and 2). Soxhlet technique gives TPC 30.05 mg GAE/g of SCSKP, TFC 4.46

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CE/g of SCSKP, IC50 value 35.89 µg/mL, catechin and gallic acid 0.04 and 9.8 mg/ g of SCSKP respectively in 6 hours of extraction. As water is the solvent, soxhlet extraction required prolonged exposure of higher temperature (100°C) at the bottom of flask where solvent evaporation takes place, and the extraction in the thimble works mostly by diffusion, i.e., no cell

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disruption to SCSKP which results in a poor yield of thermosensitive compounds. Batch extraction technique (previously optimized study) gives TPC 79.89 mg GAE/g of

SCSKP, TFC 7.29 mg CE/g of SCSKP, IC50 value 12.97 µg/mL, catechin and gallic acid 0.61 and 35.9 mg/g of SCSKP respectively when 1:15 SCSKP to water ratio, 50˚C temperature, in 105 min. of extraction. Batch extraction was performed sequentially by using the same SCSKP

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for 5 times. The yield of catechin and gallic acid in the extract obtained by every cycle was calculated (Fig. 8). Sum of yields of catechin and gallic acid got by 5 cycles of stirred batch extraction were 1.05 and 53.3 mg/ g of SCSKP, respectively in 525 min. The batch extraction process was simple, without any need for organic solvent and chemicals, but it needs more temperature, solvent, and time compared to UAE. On the contrary, deploying advance UAE for extraction of catechin and gallic acid from

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SCSKP requires less harsh conditions that avoid the degradation of essential phytoconstituents.

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This technique gives enhanced yield in lesser time compared to conventional techniques. Cavitation effect is responsible for the production and compression of bubbles, which leads to a

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rise in temperature and pressure. It causes bubbles to collapse and formation of shock waves,

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resulting in enhanced mixing. In the mechanical effect, the contact surface area between water and crude increases by allowing enhanced penetration of solvent into the SCSKP matrix. The

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thermal effect enhances the solubility of extractable phytocompounds in the solvent. Cavitation triggers the formation of small bubbles that tend to cause fast adiabatic compressions and

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expansions, which increases temperature and pressure [52]. Shear forces, sonoporation, Erosion, fragmentation, detexturation, and capillary effect are the main reported mechanisms of UAE individually or in combination [53].

TPC 100.07 mg GAE/g of SCSKP, TFC 10.11 CE/g of SCSKP, IC50 value 10.59 µg/mL,

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catechin and gallic acid 2.2 and 54.5 mg/ g of SCSKP were obtained when 1:15 SCSKP to water ratio, 35˚C temperature, power 125 W and duty cycle 60% in 12 min. of ultrasonic extraction. UAE follows the principles of the green extraction with enhanced extraction yield. Hence, UAE is superior with simple assembly over soxhlet (360 min.) and sequential batch (525 min.) techniques, which consume huge solvent, higher temperature, energy and time.

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5. Conclusion Catechin and gallic acid possess various important medicinal properties. Development of the economical, eco-friendly, more reliable and scalable process is essential to make the extraction process greener. In the present study, we have successfully intensified the extraction process of catechin and gallic acid from SCSKP by employing ultrasonic irradiation with 2.2 and 54.5 mg/g of the yield of catechin and gallic acid in 12 min at 35°C. Unlike UAE, Soxhlet and

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sequential batch extractions obtained a lesser yield of catechin, gallic acid, TPC and TFC in 360

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and 525 min. respectively. IC50 values of S. cumini extract obtained by UAE, Batch, and Soxhlet processes were 10.59, 12.97 and 35.89 mg/L, respectively. The findings from the study provide

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support for the valorization of industrial waste by using UAE SCSKP extract as a potential

Conflict of interest

Acknowledgment

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We declare no conflict of interest.

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natural antioxidant in several industries to avoid the noxious effects of allopathy.

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The authors gratefully acknowledge the University Grants Commission (UGC) India for providing financial assistance in the present research work. References [1]

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activity, total phenolic content and flavonoid concentrations of different plant parts of Teucrium polium L. subsp. polium, Acta Soc. Bot. Pol. 81 (2012) 117–122. doi:10.5586/asbp.2012.010.

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H. Boudries, N. Nabet, N. Chougui, S. Souagui, S. Loupassaki, K. Madani, K. Dimitrov, Optimization of ultrasound-assisted extraction of antioxidant phenolics from Capparis

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spinosa flower buds and LC–MS analysis, J. Food Meas. Charact. (2019). doi:10.1007/s11694-019-00144-1. [3]

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List of Figures Fig. 1: Effect of time on UAE yield of catechin and gallic acid when temperature 25°C, solute to solvent 1:20, frequency 22 kHz, power 100W, duty cycle 50%. Fig. 2: Effect of solute to solvent on UAE yield of catechin and gallic acid when temperature

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25°C, frequency 22 kHz, power 100W, time 12 min. and duty cycle 50%.

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Fig. 3: Effect of temperature on UAE yield of catechin and gallic acid when solute to solvent 1:15, frequency 22 kHz, power 100W, time 12 min. and duty cycle 50%.

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Fig. 4: Effect of power of UAE yield of catechin and gallic acid when temperature 35°C, solute

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to solvent 1:15, frequency 22 kHz, time 12 min. and duty cycle 50%.

Fig. 5: Effect of duty cycle of UAE yield of catechin and gallic acid when temperature 35°C,

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solute to solvent 1:15, frequency 22 kHz, time 12 min. and power 125W. Fig. 6: Effect of time on UAE of total flavonoid and phenolic contents (TFC and TPC) when

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temperature 35°C, solute to solvent 1:15, frequency 22 kHz, power 125W, and duty cycle 60%. Fig. 7: Effect of concentration of UAE extract vs % Antioxidant activity when time 12 min., temperature 35°C, solute to solvent 1:15, frequency 22 kHz, power 125W, duty cycle 60%. Fig. 8: Effect of sequential batch extraction on the yield of catechin and gallic acid when time

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105 min., temperature 50°C, stirring speed 250 rpm and solute to solvent 1:20.

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25

Table 1: Comparison of different techniques on extraction of catechin and gallic acid. Extraction

Time

Solvent

Temperature

Stirring

Catechin

Gallic

techniques

(min.)

(ml)

(˚C)

(rpm)

(mg/g)

acid (mg/g)

360

180

100

-

0.04

9.8

Batch

105

20

50

250

0.61

35.9

Sequential

525

100

50

250

1.05

53.3

12

15

35

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2.2

54.5

Jo

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lP

re

-p

batch UAE

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Soxhlet

25

26

Table 2: Comparison of different techniques on extraction of total phenolic contents, total flavonoid contents and IC50 values. Extraction

TPC mg GAE/ g

TFC CE/g

IC50 (PPM)

Soxhlet

30.05

4.46

35.89

Batch

79.89

7.29

12.97

UAE

100.07

10.11

of

techniques

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lP

re

-p

ro

10.59

26

27

1

35

0.9 30

of

Yield of gallic acid (mg/g)

25

0.7 0.6

20

ro

0.5

15

0.4 0.3

Catechin

0.2

5

10

15

5 0

20

25

lP

0

re

0.1 0

10

Gallic acid

-p

Yield of catechin (mg/g)

0.8

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Time (min.)

Fig. 1: Effect of time on UAE yield of catechin and gallic acid when temperature 25°C, solute to

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solvent 1:20, frequency 22 kHz, power 100W, duty cycle 50%.

27

28

Catechin

Gallic acid

1

35

0.9 30

0.6

of

20

Yield of gallic acid (mg/g)

25

0.7

0.5

15

0.4

ro

Yield of catechin (mg/g)

0.8

0.3

10

-p

0.2

0 1:05

1:10

1:15

re

0.1

1:20

1:30

1:40

5

0 1:50

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Solute: solvent

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Fig. 2: Effect of solute to solvent on UAE yield of catechin and gallic acid when temperature

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25°C, frequency 22 kHz, power 100W, time 12 min. and duty cycle 50%.

28

29

Catechin

Gallic acid

1.8

50

1.6

45

of

30

1

25

ro

0.8

20

0.6

15

-p

Yield of catechin (mg/g)

35

1.2

Yield of gallic acid (mg/g)

40

1.4

0.4

0 25

30

re

0.2

35

45

10 5 0

55

65

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Temperture (˚C)

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Fig. 3: Effect of temperature on UAE yield of catechin and gallic acid when solute to solvent

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1:15, frequency 22 kHz, power 100W, time 12 min. and duty cycle 50%.

29

30

Catechin

Gallic acid

2.5

50 45

35 30

of

1.5

Yield of gallic acid (mg/g)

40

ro

25

1

20 15

-p

Yield of catechin (mg/g)

2

0 44

79

re

0.5

102

125

168

10 5 0 215

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Power (W)

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Fig. 4: Effect of power of UAE yield of catechin and gallic acid when temperature 35°C, solute

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to solvent 1:15, frequency 22 kHz, time 12 min. and duty cycle 50%.

30

31

Catechin

Gallic acid

2.5

70

60

1.5

of

40

Yield of gallic acid (mg/g)

50

ro

30

1

20

-p

Yield of catechin (mg/g)

2

0 10

20

40

re

0.5

50

60

10

0 80

100

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Duty cycle (%)

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Fig. 5: Effect of duty cycle of UAE yield of catechin and gallic acid when temperature 35°C,

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solute to solvent 1:15, frequency 22 kHz, time 12 min. and power 125 W.

31

32

12

120

100

8

80

6

60

of

10

TPC mg GAE/g of SCSKP

TPC

4

40

ro

TFC mg CE/g of SCSKP

TFC

20

0 2

4

6

8

10

12

re

0

-p

2

14

16

0 18

lP

Time (min.)

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Fig. 6: Effect of time on UAE of total flavonoid and phenolic contents (TFC and TPC) when

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temperature 35°C, solute to solvent 1:15, frequency 22 kHz, power 125W, and duty cycle 60%.

32

33

100 90

Catechin Gallic acid extract obtained from ultrasound assisted technique

80

of

60 50 40

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% Antioxidant activity

70

-p

30 20

0 25

50 75 Concentration (ppm)

100

lP

10

re

10

Fig. 7: Effect of concentration of UAE extract vs % Antioxidant activity when time 12 min.,

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temperature 35°C, solute to solvent 1:15, frequency 22 kHz, power 125W, duty cycle 60%.

33

34

60 Gallic acid 50

0.8

40

0.6

30

of

Yield of catechin (mg/g)

Catechin 1

Yield of gallic acid mg (mg/g)

1.2

20

ro

0.4

10

0 1st stage

2nd stage

3rd stage

4th stage

0

5th stage

lP

re

total yield

-p

0.2

Fig. 8: Effect of sequential batch extraction on the yield of catechin and gallic acid when time

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105 min., temperature 50°C, stirring speed 250 rpm and solute to solvent 1:20.

34