Microwave Assisted Batch Extraction of Polyphenols From Sea Buckthorn Leaves

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Chemical Engineering Communications

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Microwave Assisted Batch Extraction of Polyphenols From Sea Buckthorn Leaves

Ioana Asofiei, Ioan Calinescu, Adrian Trifan, Iulia Gabriela David & AdinaIonuta Gavrila

To cite this article: Ioana Asofiei, Ioan Calinescu, Adrian Trifan, Iulia Gabriela David & AdinaIonuta Gavrila (2016): Microwave Assisted Batch Extraction of Polyphenols From Sea Buckthorn

Leaves, Chemical Engineering Communications, DOI: 10.1080/00986445.2015.1134518

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Microwave Assisted Batch Extraction of Polyphenols from Sea Buckthorn Leaves

Ioana Asofiei1, Ioan Calinescu

1, Adrian Trifan

1, Iulia Gabriela David

2, Adina Ionuta

Gavrila1

1

Department of Bioresources and Polymer Science, University Politehnica of Bucharest,1-7 Gh. Polizu, Zip Code 011061, Bucharest, Romania,2Department of Analytical

Chemistry, University of Bucharest, 90 Panduri, Zip Code 050657, Bucharest, Romania

Corresponding author to Adina Ionuta Gavrila, Department of Bioresources and PolymerScience, University Politehnica of Bucharest, 1-7 Gh. Polizu, Zip Code 011061,

Bucharest, Romania. E-mail: [email protected]

Abstract

Extraction of polyphenols from Sea Buckthorn leaves using microwave assisted

extraction (MAE) is described. The influence of different parameters on the extraction

process (reactor type, stirring rate, extraction time, temperature, ethanol/water ratio) was

studied. The polyphenolic extracts were analyzed in order to determine the total phenolic

content (TPC) by Folin-Ciocalteu method or by differential pulse voltammetry (DPV)

and the concentration of the main polyphenolic compounds by HPLC. The specific

microwave energy was also determined. MAE leads to a shorter extraction time (7.5 min

versus 30 min for the conventional method). The best results of MAE were obtained at a

temperature of 90°C, using a solvent/plant ratio of 20/1 and 50% ethanol in the extraction

solvent. The highest values of antioxidant capacity were obtained for polyphenolic

extracts resulted from microwave extraction.

KEYWORDS: microwave assisted extraction, polyphenols, Sea Buckthorn

INTRODUCTION


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Hippophae rhamnoides L. known as Sea Buckthorn is a nitrogen fixing bush widely

found in temperate and subtropical zones. All parts of the shrub are rich in bioactive

compounds. Recent developments have shown the beneficial properties of Sea Buckthorn

leaves extracts. The leaves contain polyphenolic compounds such as ferulic acid, caffeic

acid, flavonols, gallic acid, kaempferol, isorhamnetin, epigallocatechin, epicatechin, and

gallocatechin. The high content of polyphenols in plant leaves have shown antioxidant

properties and can be used to prevent the damaging effect of oxidant radicals

(Upadhyayet al., 2010; Kant et al., 2012). These polyphenols present pharmacological

activities such as radioprotective, immunomodulatory, anti-inflammatory and tissue

regeneration (Chawla et al., 2007; Suryakumaret al., 2011).

Extraction of bioactive compounds from vegetable materials is determined by different

factors such as extraction yield, production cost and uses of safety and environmental

friendly solvents (Desai et al., 2010). The most common extraction methods are Soxhlet

extraction, conventional extraction and heat reflux extraction. These methods require

long extraction times, expensive organic solvents, relatively high solvent consumption

and high temperatures that could cause the degradation of valuable constituents.

Recent developments present more advantageous techniques such as pressurized fluid

extraction (PFE), ultrasound assisted extraction (UAE), pulsed electric field (PEF),

supercritical fluid extraction (SFE) microwave hydro-diffusion and gravity (MHG) and

microwave assisted extraction (MAE).


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Extraction of bioactive compounds by PFE technique is carried out under high

temperature and pressure. This method is unhelpful for polyphenols which are heat

sensitive (Kronholm et al., 2007). In UAE of polyphenols from plants the rupture of

matrix is initiated by ultrasonic irradiation. By sonication the solvent penetrates the plant

cells due to the jet generated by the bubble breakdown. This jet enlarges the cell pores

and act as a micro-pump, forcing the solvent to enter into the cells and dissolve the

components (Mason et al., 2011). Thus, the active components rapidly diffuse from plant

tissue to solvent. UAE method is useful for thermolabile compounds due to the short

extraction time. The extraction efficiency is influenced by the US power which is lower

for longer extraction time (Pasrija et al., 2015).

PEF treatment increases the permeability of plant tissues. The cell membranes are

charged and pores are formed in the membranes facilitating extraction of cellular fluid.

PEF has been used to extract polyphenols. The PEF efficiency was improved when the

treatment was performed in the presence of ethanol. PEF pre-treatment increased both the

extraction kinetics and the maximum yield of polyphenols (Chemat et al., 2015).

The SFE technique using CO2 as a solvent has the advantages of mild extraction

conditions (short extraction time and low extraction temperature), but CO2 is non-polar

and this could limit its use in the extraction of polar polyphenols (Temelli et al., 2009).

MAE is a promising technology to extract bioactive compounds from vegetal material by

combination of extraction technique with microwave heating. The microwave heating is

based on the effect of the microwaves on material molecules by ionic conduction and

dipole rotation. Thus, an efficient heating requires either polar extraction solvents or


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polar materials (Desai et al., 2010; Lew et al., 2002). Recently, MAE has been

extensively employed for the extraction of polyphenols from various parts of plants due

to its benefits such as shorter extraction time, good control of heating process, high

extraction efficiency and selectivity, better extraction yield and reduced quantity of

energy (Périno-Issartier et al., 2010, Chemat et al., 2015, Filly et al., 2014, Li et al., 2013,

Xiao et al., 2008; Rostagno et al., 2007; Chen et al., 2007; Spigno et al., 2009; Wanget

al., 2010; Alupului et al., 2009; Calinescu et al., 2014; Calinescu et al., 2002).

MHG is a green extraction technique that combines microwave heating and gravity at

atmospheric pressure. MHG is a suitable method for laboratory and industrial-scale

applications (extraction of pigments, essential oils, and antioxidants from plants).

Extraction of polyphenolic compounds from onion and Sea Buckthorn by MHG led to a

shorter extraction time, no solvent used, and polyphenolic extract showed a higher

phenolic content with greater antioxidant activity in comparison to classical extraction

methods (Li et al., 2013).

The purpose of this paper is to establish the optimal conditions for polyphenols extraction

from Sea Buckthorn leaves using batch microwave assisted technique. The influence of

different extraction parameters such as reactor type, stirring rate, extraction temperature

and time, solvent to plant material ratio and solvent concentration have been studied.

MATERIALS AND METHODS

A. Materi als


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The Sea Buckthorn leaves ( Hippophae Rhamnoides L.) were harvested in the summer of

2014 at Hofigal S.A. in Furculesti. The fresh leaves were dried in air flow-heating oven at

60°C to a constant weight. The dried leaves were ground using an electric grinder and

sieved to a particle size under 0.5 mm. The ground Sea Buckthorn leaves were dosed in

samples of 25 g (in sealed plastic vessel) and stored at 4-5°C until they were used for

extraction of the phenolic compounds.

Folin-Ciocalteu reagent (Merck), ethanol and sodium carbonate were analytical grade.

The standard used for TPC analysis was gallic acid from Sigma-Aldrich. For the HPLC

quantification of phenolic compounds, the following standards were used: gallic acid,

caffeic acid, chlorogenic acid, catechin, ferulic acid, p-coumaric acid and rutin from

Sigma-Aldrich.

B. Polyphenols Extraction Procedure

The MAE of phenolic compounds was performed using a microwave system (Biotage

Initiator). The conventional extraction of polyphenolic compounds was carried out in a

water bath using a heating plate equipped with a temperature controller unit and magnetic

stirring. The extractions were performed in triplicate, using a 20:1 ratio of solvent to

plant. The experiments were carried out at different temperatures (60°C, 90°C and

120°C) and different stirring rates (300 and 900 rpm). The extractions occur in a mixture

of ethanol in water using a concentration of 0%, 25% and 50% ethanol in water.

Individual experiments were performed considering the following extraction times: 50,

100, 150, 200, 300, 450, 600, 900 and 1800s. After the extraction, the mixture was


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centrifuged at 3000 rpm for 5 min at room temperature and the supernatant was collected

and fresh analyzed every time.

C. Anal ysis

Determination Of Total Phenolic Content

Total phenolic content of extracts was determined colorimetrically using the Folin-

Ciocalteu method according to International Standard ISO 14502-1 with minor

modifications. The fresh extracts were diluted 125 times with distilled water. Further, 0.5

mL of diluted extract was mixed with 5 mL of 10% Folin-Ciocalteu reagent and stirred

for 5 min to perform the reaction. Next, 1.5 mL of 20% Na2CO3·10H2O and 3 mL of

distilled water were added. Before analysis, the samples were kept for 30 min in the dark

at room temperature. The absorbance was measured at 760 nm using a Shimadzu UV

mini-1240 UV/Visible Scanning Spectrophotometer. The samples were analyzed in

duplicates. The results were quantified as milligram of gallic acid equivalents per 1 gram

of dry matter (mg GAE/g DM) using standard curve prepared for gallic acid solutions (1-

5 mg/mL).

DPV Determination Of Total Phenolic Content

Voltammetric measurements were carried out using an electrochemical system

(potentiostat/galvanostat) AUTOLAB PGSTAT 12. The system was equipped with a

voltammetric cell consisting of a working electrode (pencil graphite electrode - PGE)

(David et al., 2015), an Ag/AgCl (3M KCI) reference electrode and a platinum wire as

auxiliary electrode. The optimized parameters for the differential pulse voltammetric


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(DPV) measurements were the following: initial potential + 0.200 V, final potential +

1.000 V, scan rate v=50 mV/s, pulse period PP=180 msec, pulse width PW=60 msec,

sample width SW=10 msec, pulse amplitude PA= E=20 mV.

Differential pulse voltammograms (DPVs) were recorded for 10 mL of 1000 fold diluted

extract samples. 0.1 M H2SO4 was used as supporting electrolyte. If the potential applied

to the PGE was scaned towards anodic values, the polyphenolic compounds from plant

extracts are oxidized. The DPVs of all samples showed an anodic peak at potentials

higher than 500 mV. The intensity of this peak was proportional to the amount of total

polyphenols contained in the analysed extract. Increasing the dilution leads to a more

pronounced peak. At 1.000 times dilutions, the peak current varied linearly with the gallic

acid (GA) concentration added in the sample as standard.

In order to determine the TPC of extracts the standard addition method was used. Thus,

for each sample DPVs were recorded before and after each of the three additions of 0.1

mL GA stock solution (2.94∙10-3

M) (Figure 1A). The TPC of samples was calculated

using the equation of the I pa = f (Cad) curve where I pa represents the maximum peak

intensity and Cad the concentration of added GA (Figure 1B).

HPLC Analysis Of Major Phenolic Compounds

The polyphenolic compounds extracted were further analyzed by HPLC analysis. The

analyses were undertaken with the help of a Jasco HPLC. The system includes: UV-2075

detector; PU-2080 plus pump; LG-2080_4 gradient unit; DG-2080_4 degasser;


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Teknokroma Nucleosil 100 C18 (10micro m, 250x0.4) separation column. Analyses were

performed at a flow rate of 0.5 mL/min using water with 2% v/v acetic acid (solvent A)

and methanol (solvent B) under the following gradient program: 0-9 min 70% A and 30%

B, 9-18 min 60% A and 40% B, 18-30 min 50% A and 50% B, 30-60 min 50% A and

50% B and then returned to initial condition for a 10 min reequilibration, with total run

time 70 min. The analytes were detected at 270 nm. For better separation of polyphenols

they were extracted from the ethanolic solution resulted from extraction. Extraction of

polyphenols from ethanolic solvent was performed using ethyl ether as solvent (50%

polyphenols solution and 50% ethyl ether are stirred 10 min at room temperature).

Samples of polyphenols in ethyl ether have been used for HPLC analysis. Analytes were

identified by comparison of retention times with known standards.

RESULTS AND DISCUSSION

1. Factors That Influence The MAE Of Polyphenolic Compounds From Sea

Buckthorn Leaves

A) I nf luence Of The Stir ri ng Rate On The Microwave Polyphenols Extraction

Polyphenols extraction from Sea Buckthorn leaves is a complex process influenced by a

very good contact between solvent and plant material. Therefore, in order to allow the

diffusion of the solvent in plant tissue and an efficient extraction of bioactive compounds,

intensely mixture stirring is required. One of the studied parameters was the influence of

stirring rate on the extraction of polyphenolic compounds. Extraction of polyphenols was

carried out at different stirring rates (300 and 900 rpm) and different extraction times.

The results are shown in Figure 2.


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As shown in figure 2, the increase of the stirring rate, from 300 rpm to 900 rpm, led to a

three fold increase in the total phenolic content. In addition, at a stirring rate of 300 rpm,

a longer extraction time determines a slow increase of TPC. For this reason, a low stirring

rate requires very long extraction times in order to achieve an efficient extraction.

Moreover, it is noted that for higher stirring rate (900 rpm) a longer extraction time (over

200 s) leads to a slight decrease in TPC.

B) I nf luence Of The Reactor Type On The Microwave Batch Extraction Of

Polyphenols

The extraction process is more effective if the stirring rate is higher. A method of

improving the mixing of the extraction medium is to change the geometry of the reactor.

Therefore, the reactor was modified in order to allow the use of a bigger magnetic stirrer.

For this study, extraction of polyphenolic compounds was carried out using two types of

reactors (normal Biotage reactor - 1 and a modified reactor – 2, as shown in Figure S1).

The two reactors make possible the extraction of polyphenols using the same volume of

solvent (5 mL). Thus, the microwave extractions were performed at various stirring rates

using these two types of reactors. The results of the influence of the reactor type on the

polyphenols extraction are shown in Figure 3.

Changing the geometry of the reactor and using a larger stirrer improved the extraction

process of polyphenols, as shown in Figure 3. Although, at short extraction times, the

polyphenols concentration is about the same for both reactors, at longer times (after 200

s) the TPC is significantly higher (around 20%) for modified reactor (Fig. S1, reactor 2).


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Given the fact that the highest amounts of polyphenols were obtained for reactor 2, the

following studies of polyphenols extraction from Sea Buckthorn leaves were carried out

using this reactor.

C) I nf luence Of E thanol Concentration On The Extraction Of Polyphenols

The most common method of extraction of polyphenols from plants involves polar

organic solvents that destroy the cell membrane and dissolve the polyphenols. Most

organic solvents are volatile, flammable, and often toxic and are responsible for the

greenhouse effect and environmental pollution. The increasing demand of green

extraction, using natural products and extracts, made us use a less toxic polar bio-solvent

as ethanol. Even if it is potentially explosive and inflammable, ethanol is used on a large

scale due to its low price, biodegradability and availability in high purity (Chemat et al.,

2012). This solvent is considered a natural solvent (Chemat et al., 2015) that can be

produced from agricultural sources – sugar beet and cereals. The study of the effect of

solvent on the extraction process was achieved by using different concentrations of

ethanol in water (Figure 4).

The increase of the concentration of ethanol in the extraction solvent mixture increases

the TPC, as shown in Figure 4. Thus, the use of a 25% ethanol concentration in water

results in achieving polyphenol concentrations of about 90 mgGAE/g DM, higher than

when using only water as solvent. Moreover, increasing the concentration of ethanol in

water to 50% doubles the amount of extracted polyphenols compared to the extraction

without ethanol. This behavior can be explained by the difference in solubility of


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compounds in water and ethanol. The organic solvent (ethanol) dissolves cell membranes

and water causes the swelling of the vegetable material, thus allowing the solvent to

penetrate more easily into the solid matrix. Thereby, the extractability of polyphenols

increases. Therefore, the choice of the solvent / water ratio depends on the composition of

the polyphenols. In conclusion, a concentration of 50% ethanol in water results in an

efficient extraction of polyphenols from Sea Buckthorn leaves at a temperature of 60 °C.

D) I nf luence Of Temperature And Extraction T ime On The Polyphenols Extraction

Another studied parameter on polyphenols extraction from Sea Buckthorn leaves is the

effect of temperature on the TPC. The temperature is an important factor in the solid-

liquid extraction process. The increase of the temperature increases the solubility of the

polyphenols and also increases the mass-transfer coefficient between the matrix material

and extraction medium. Thus, the experiments were carried out at different temperatures.

The extraction of phenolic compounds is also influenced by the extraction time, which

can affect the solubility of polyphenols. A long extraction time can lead to the oxidation

of polyphenols or can change the conformation of extracted polyphenols.

The microwave power is an important factor for microwave assisted extraction of

bioactive compounds from plants. For each experiment, the microwave power and

temperature profiles have been recorded. Using the above mentioned recorded values, we

determined average microwave power during heating and extraction, and the microwave

energy supplied to the system (Equations S1-S7, Table SI). The specific energy was

determined by the following equation:


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total sp

E J E

TPC mgGAE (1)

Where: Esp - specific energy [J/mg GAE]; Etotal – total energy [J]; TPC – total phenolic

content [mg GAE/g DM].

Figure 5 shows the influence of extraction time and temperature on the TPC and specific

energy by batch MAE from Sea Buckthorn leaves.

A higher temperature increases the polyphenols content. This can be explained by the

increase of polyphenols solubility and a high value of mass-transfer coefficient between

the matrix and the extraction medium at high temperatures. In addition, a high extraction

temperature results in the destruction of the plant cell walls. Thus, this can lead to a

further better penetration of the solvent in the cell walls, thus allowing the dissolution of

polyphenols in the extract. Likewise, Figure 5 shows that at high temperature (120⁰C) the

maximum TPC is obtained faster than for lower temperatures (100 s for 120⁰C and 450 s

for 60⁰C and 90⁰C). However, the highest concentration of polyphenols was obtained for

the extraction temperature of 90⁰C. After exceeding the maximum, at a temperature of

120 ⁰ C the TPC decreases much faster than the extractions at 60⁰ C or 90⁰ C. This can

be explained by the degradation of polyphenols at high temperatures. From these data it

was concluded that the optimal temperature for MAE of polyphenols from Sea Buckthorn

leaves is 90⁰C.

As is shown in figure 5, the lowest specific energy corresponds to the experiments carried

out at 60⁰ C. At 90⁰ C, slightly higher values of specific energy are obtained. In addition,


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the amount of extracted polyphenols at 90⁰ C is higher than the amount obtained for

other extractions (60⁰ C and 120⁰ C). For this reason, the extraction at 90⁰ C can be

considered more efficient from TPC and specific energy points of view.

These values of specific energy are expected to decrease when scaling up. This effect is

typical for pilot and industrial installations where a higher ratio between the microwave

energy absorbed by the system and the one delivered is required. Reactors with small

volumes are used in the experiments presented in this work and for this reason, the ratio

between the required and total energy is only 1 to 4 (Table SI).

2. Microwave Assisted Extraction Vs. Conventional Extraction

To highlight the effect of microwaves on polyphenols extraction from Sea Buckthorn

leaves, experiments were carried out under the same conditions, for various temperatures,

both by microwave assisted extraction and conventional extraction (Figure 6).

The increase of the extraction time leads to an increase in the total phenolic content,

reaching a maximum after 7.5 min in the MAE and after 30 min in the case of

conventional extraction. However, a further increase of the extraction time leads to the

decrease of the polyphenolic content. Therefore, although the maximum concentration of

polyphenols is about the same for both methods, the MAE leads to reducing the

extraction time.

3. Total Phenolic Content Determination By Differential Pulse Voltammetry


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Differential Pulse Voltammetry (DPV) can measure the ability of compounds to donate

electrons. This can be related to the antioxidant capacity of polyphenolic compounds. For

lower values of the oxidation peak potential, the oxidation occurs easily. Therefore, the

antioxidant capacity of the compound is higher (Abou Samra et al., 2011).

Table I presents a comparison between the results of the TPC analysis performed by the

Folin-Ciocalteu method and by DPV. One can observe that the DPV values are lower

than those obtained applying the Folin-Ciocalteu method. This can be explained by the

fact that the Folin-Ciocalteu reagent is less selective and can be also reduced by other

non-phenolic compounds present in the analysed samples (Seruga et al., 2011).

4. Polyphenols Composition

TPC values can be influenced by a number of other compounds with reducing character

that are extracted from plant (as shown by comparing TPC values obtained by Folin

Ciocalteu and DPV methods). For this reason, the polyphenolic extracts were analyzed by

HPLC analysis to quantify the content of polyphenols (Table II). The phenolic

compounds analyzed by HPLC depended on the extraction method. The main

components from Sea Buckthorn leaves extracts were gallic acid, catechin, caffeic acid

and rutin (Bittova et al., 2014). Catechin was the predominant polyphenolic species,

followed by gallic acid and rutin.


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From the results shown in Table II it can be noticed that the increase of extraction time

led to the increase of total phenolic content (TPC) values, but the major components

content slightly decreased at long extraction time.

CONCLUSIONS

The aim of this work was to determine the best conditions for MAE of polyphenols from

Sea Buckthorn leaves. The influence of various parameters was determined: stirring rate,

extraction time, type of reactor, concentration of ethanol in water and temperature. MAE

leads to reduction of the extraction time compared to conventional extraction. Increasing

the temperature leads to higher TPC, but at long extraction time concentration of

polyphenols decreases. This can be explained by the degradation of polyphenols. A

higher stirring rate increases the polyphenols content, but another important factor in

order to obtain an efficient stirring of the extraction medium is the geometry of the

reactor. Total phenolic content increases with the increase of the concentration of ethanol

in water. However, the ratio of the solvent is closely related with the solubility of

polyphenolic compounds found in Sea Buckthorn leaves.

The effect of heating time and specific energy on the total polyphenolic content was

investigated. In comparison with other extraction temperatures, the experiments carried

out at 90 °C led to a higher TPC obtained with a lower value of specific energy.

DPV analysis was used as an alternative method for TPC determination. The values

obtained by DPV are lower than those resulted by Folin-Ciocalteu method, due to the fact


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that Folin-Ciocalteu reagent is less selective and can be also reduced by other non-

phenolic compounds. This low selectivity was confirmed by the HPLC analysis.

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Table 1.

No. Experimental conditions TPC [mg

GAE/g DM]

Ethanol

concentration,[%]

Extraction

time, [s]

Heating

type

Folin-

Ciocalteumethod

DPV

method

1 50 450 Microwave 134.96 93.71

2 25 450 Microwave 95.02 70.34

3 0 450 Microwave 66.00 47.90

4 0 150 Microwave 62.13 41.40

5 50 450 Conventional 123.93 86.49


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Table 2.

No. Temperature,

[°C]

Time,

[s]

Compound [mg/g DM] TPC,

[mg

GAE/g

DM]

Gallic

acid

Catechin Caffeic

acid

Rutin

1. 90 50 3.24 9.37 1.51 3.94 109.75

2. 90 100 3.34 10.09 1.64 3.79 114.84

3. 90 200 3.31 9.24 1.44 4.02 125.31

4. 90 300 3.13 9.02 1.4 3.01 131.14


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.

Microwave Assisted Batch Extraction of Polyphenols From Sea Buckthorn Leaves

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