Optimization of ultrasound-assisted extraction of isogentisin, gentiopicroside and total polyphenols from gentian root using response-surface methodology

Industrial Crops & Products 139 (2019) 111567

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Optimization of ultrasound-assisted extraction of isogentisin, gentiopicroside and total polyphenols from gentian root using responsesurface methodology

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Jelena Živković , Teodora Janković, Nebojša Menković, Katarina Šavikin Institute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000, Belgrade, Serbia

A R T I C LE I N FO

A B S T R A C T

Keywords: Gentian Iridoid Xanthone Extraction parameters

In this study we have established optimal conditions for ultrasound-assisted extraction (UAE) of isogentisin, gentiopicroside and total polyphenols from Gentiana lutea root using response surface methodology (RSM) based on central composite design (CCD). The influence of extraction time (X1: 5–65 min), ethanol concentration (X2: 10–90%), solid to solvent ratio (X3: 1:10–1:50) and extraction temperature (X4: 20–80 °C) was analysed. The optimal conditions of extraction procedure were following: X1: 31 min, X2: 49%, X3: 1:42, and X4: 80 °C. Experimentally achieved values were in accordance with those estimated by RSM model, suggesting applicability of the utilized model and the favorable result of RSM application in optimization of extraction parameters.

1. Introduction Gentiana L. is a globally distributed genus which includes around 400 species appeared in Asia, Europe and America. One of the well known species is Yellow Gentian, Gentiana lutea L., showing favorable properties in stomach, liver and gall ailments (Blumenthal, 1998). G. lutea is a widely distributed in mountain regions located in south and central Europe, growing wildly in Spain, France, and Balkan countries. In Europe, it is below wildlife protection and it is cultivated dominantly in France and Germany (European Medicines Agency, 2009). Root and rhizoma (Gentianae radix) are officinal in European as well as in many nationals Pharmacopoeias (European Pharmacopoeia, 2014; Pharmacopoeia Jugoslavica, 1984). It is also very popular in traditional medicine of many countries, including Serbia, especially among mountain inhabitants. It is used to promote appetite and to enhance digestion (European Medicines Agency, 2009). The fermented roots are also applied for the manufacturing of bitter alcoholic beverages (Mustafa et al., 2016). Underground parts are characterized by diverse chemical composition. The bitter taste originates from the main constituents, secoiridoids. Among them, the most abundant are gentiopicroside, swertiamarin and sweroside. The roots are also presented with biphenyl derivatives amarogentin, amaropanin and amaroswerin (Wagner et al., 1984). Other groups of pharmacologically active compounds comprise xanthones (gentisin, isogentisin, gentioside) and C-glucoflavones

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(isoorientin, isovitexin), (Šavikin et al, 2010). Due to such chemical composition, the underground parts of G. lutea possesses lot of biological properties in particular cholagogue, antimicrobial, anthelmintic, radioprotective, anti-atherosclerotic, neuritogenic activity (Öztütk et al., 1998; Menković et al., 1999, 2010; Pontus et al., 2006; Mustafa et al., 2015a). Chemical composition of Gentiana lutea root extract has been extensively studied (Amakura et al., 2016; Mustafa et al., 2015b), and several papers examined the effects of different solvents on the extraction efficiency (Ariño et al., 1997; Kušar et al., 2010). However, these studies used classical optimization approach when one-factor-ata-time was used, and there are no reports related to the optimization of conditions for extraction of its constituents using response surface methodology (RSM). The composition of final extract can be influenced by numerous factors in particular extraction time, ethanol concentration, solid to solvent ratio (SS ratio) and extraction temperature (Hammi et al., 2016). Main disadvantages of traditional extraction techniques (Soxhlet extraction or maceration) are long extraction time and application of immense quantities of solvent (Albuquerque et al., 2017; Bimakr et al., 2011). Today various techniques such as ultrasound-assisted extraction are used to increase efficancy of extraction process and quality of final product (Oliveira et al., 2016). In comparision with conventional techniques ultrasound assisted extraction reduces extraction time and energy consumption (Upadhyay et al., 2015). For higher extraction of different compounds different

Corresponding author at: Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000, Belgrade, Serbia. E-mail address: [email protected] (J. Živković).

https://doi.org/10.1016/j.indcrop.2019.111567 Received 5 January 2019; Received in revised form 18 June 2019; Accepted 13 July 2019 0926-6690/ © 2019 Published by Elsevier B.V.

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linear, β11, β22, β33 and β44 - quadratic and β12, β13, β14, β23 and β24 interaction regression coefficient terms, respectively. Optimal extraction parameters were calculated taking into account following responses: total phenolics content (TPC), as well as isogentisin and gentiopicroside amounts. Design of experiment, analysis of data and determination of optimal conditions were performed using STATGRAPHIC Centurion XVII software (Statpoint Technologies, Inc., USA). Significance of independent variables and interactions among them was evaluated using ANOVA and 0.05 was set as significance level value. Standardized Pareto charts have been used to analyze significance of effect of investigated variables on refered responses. The efficiency of the model was estimated using the coefficient of determination (R2), as well as p values for the model and lack-of-fit testing. Confirmation of the model was performed by implementing achieved optimal conditions (extraction time, SS ratio, ethanol concentration and extraction temperature) for UAE aiming to achieve the greatest TPC, isogentisin and gentiopicroside amounts. Confirmation analysis was conducted in triplicate. At the end of investigation, optimal parameters for the UAE extraction of maximal content of all analysed responses simultaneously (TPC as well as amounts of individual compounds isogentisin and gentiopicroside) were determined by employing the desirability function method.

extraction conditions are favourable. That is why optimization of process parameters is required in order to prepare G. lutea root extract with maximal content of iridoid and polyphenolic compounds. RSM represents valuable tool in analysis of interactions between factors and it can be used for quantitative characterization of the effects of selected parametres on estimated responses (Yuan et al., 2015). It is more suitable compared to one factor at a time optimization that has been traditionally used, since it lessens space, time and material usage (Lee et al., 2012). Aiming to optimize extraction process, RSM can be applied as a valuable statistical tool. In our investigation we have used RSM based on central composite design (CCD) in order to establish the impact of extraction time, ethanol concentration, SS ratio and extraction temperature on total polyphenols content (TPC) as well as on the amount of isogentisin (as xanthone compound) and gentiopicroside (as secoiridoid compound) from G. lutea root. 2. Methodology 2.1. Plant material Gentian root was purchased from the pharmacy store at the Institute for Medicinal Plants Research „Dr. Josif Pančić “from Belgrade, Serbia (batch: 24630915). Plant originated from Mt. Tara in Southwest Serbia. It was harvested in 2014 and packed in paperbags in warehouse conditions prior to analysis. Before the analysis, pieces of the roots were grounded using a laboratory mill (IKA A11 basic, IKA®-Werke GmbH & Co. KG, Germany). Particles from 0.75 to 2 mm size were separate using laboratory sieves according to Yugoslav Pharmacopoeia (Ph. Yug. V) and were further used for the optimization of the extraction.

2.3. UAE methodology Ultrasound-assisted extraction was performed in an ultrasonic bath (bath power 35 W, continuous mode at frequency of 40 kHz, Maget, Bela Palanka, Serbia). Pulverized gentian root (0.5–2.5 g) was combined with 25 mL of ethanol at different concentrations (10–90%) and with various SS ratios (1:10–1:50). The extraction was performed at diverse temperatures (20–80 °C) for varying time periods (5–65 min) and the position of the extrcat in Erlenmeyer flask (100 mL) was 5 cm below the water surface in the bath. After process completion the obtained samples were filtered through filter paper and until further investigation they were kept at -18 °C.

2.2. Applied experimental design and statistical model CCD has been applied for the optimization of UAE of gentian root as published in our earlier study (Živković et al., 2018).The design was structured from 30 randomly assigned runs with five central pont replicates. Four variables were selected as the responses in the planned experiment, and each of them was analysed at five various levels. Values of independent variables (both natural and coded) that were used in CCD are provided in Table 1. Following equation was applied for relationship between coded and real values of variables:

2.4. Spectrophotometric determination of TPC Previously published Folin-Ciocalteu method was applied for spectrophotometric determination of TPC in G. lutea samples (Waterman and Mole, 1994). All experiments were conducted in triplicate.

Xv =(xi-x0)/Δxi

2.5. HPLC analysis

Xv represent coded value of the independent variable, xi represents the actual value of the independent variable, x0 is the actual value of the independent variable in the center of the domain and Δxi is the step change value. Suggested model for every response (Y) was with linear, quadratic and interactive components:

HPLC-DAD analysis of gentiopicroside and isogentisin was performed according to formerly reported method (Balijagić et al., 2012). Calibration curves were applied for calculation of the amounts of investigated compounds. The results are provided as mg/g of dry weight.

Y = β0 + β1X1 + β2X2 +β3X3 + β4X+ β11X12 + β22X22 + β33X32 + β44X42 + β12X1X2 + β13X1X3 + β14X1X4 + β23X2X3 + β24X2X4

3. Results and discussion 3.1. Model adequacy

where Y is response, X1 - extraction time, X2 - ethanol concentration, X3 - the SS ratio, X4 - extraction temperature, βo – intercept, β1, β2, β3 -

In our investigation research we have employed a four factor, five level, CCD aiming to achieve extract with maximum content of gentiopicroside (as secoiridoid compound), isogentisin (as xanthone compound) and total polyphenolic content. Several papers examined the effects of different solvents on the extraction efficiency of Gentiana lutea root (Ariño et al., 1997; Kušar et al., 2010). However, these studies used classical optimization approach when one-factor-at-a-time was used, and there are no reports related to the optimization of conditions for extraction of its constituents using response surface methodology (RSM). Experimental results achieved for previously mentioned responses (TPC, content of isogentisin and gentiopicroside) using various

Table 1 Real and coded values of UAE parameters. Independet variable

Time (min) EtOH concentration (%) SS ratio (1:X3) Temperature (°C)

Variables with their coded levels

X1 X2 X3 X4

−2

−1

0

1

2

5 10 10 20

20 30 20 35

35 50 30 50

50 70 40 65

65 90 50 80

2

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Table 2 Central composite design with coded parameters of UAE, and experimentally observed responses in the obtained extracts. Run

Extraction time - X1 (min)

EtOH concentration - X2 (%)

SS ratio - X3 (1:X3)

Extraction temperature X4 (ºC)

TPC (mg GAE/ g dw)

Gentiopicroside (mg/g dw)

Isogentisin content (mg/ g dw)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

0 −1 1 −1 1 1 −1 −1 1 0 1 −1 1 −1 −1 1 0 −1 0 1 0 2 0 −2 0 0 0 0 0 0

0 −1 1 1 1 −1 1 −1 −1 0 −1 −1 −1 −1 1 1 0 1 0 1 0 0 0 0 0 0 2 −2 0 0

0 −1 −1 1 1 1 −1 1 −1 0 −1 1 1 −1 −1 1 0 1 0 −1 0 0 0 0 2 0 0 0 0 −2

0 1 1 1 −1 1 −1 −1 −1 0 1 1 −1 −1 1 1 0 −1 0 −1 0 0 2 0 0 −2 0 0 0 0

30.20 21.00 24.70 28.20 27.90 30.30 19.90 29.30 26.70 38.30 28.80 35.00 29.90 29.80 23.80 32.90 32.40 26.20 31.70 24.50 32.40 32.90 33.90 32.30 35.60 35.70 18.00 23.30 31.30 23.00

21.19 21.34 21.53 21.88 21.11 20.92 21.52 20.41 21.05 22.11 21.81 20.67 21.85 19.27 20.79 20.98 21.73 20.60 21.72 21.09 21.64 21.39 22.92 22.18 20.50 21.19 18.47 20.70 21.80 19.94

4.19 4.00 4.13 4.31 4.05 4.27 3.88 3.47 3.29 4.49 4.22 4.11 3.85 2.96 4.04 4.28 4.24 3.92 4.28 3.88 4.31 4.43 4.93 4.31 4.26 4.17 3.06 2.48 4.26 3.85

TPC – total polyphenols content; dw – dry weight.

3.2. Influence of extraction variables on TPC

Table 3 Analysis of the variance of the fitted second-order polynomial models. Isogentisin

Gentiopicroside

Source

Total polyphenolic content p-value*

Model X1-extraction time X2-EtOH concentration X3-ss ratio X4-temperature X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 X12 X22 X32 X42 Lack of fit R2

0.0001 0.3222 0.0265 0.0003 0.6129 0.3175 0.4802 0.6596 0.7121 0.3013 0.1723 0.5155 0.0000 0.0417 0.7018 0.7892 0.82

0.0000 0.0200 0.0000 0.0004 0.0000 0.0769 1.0000 0.4086 0.2226 0.0009 0.1589 0.5026 0.0000 0.0141 0.0181 0.5505 0.98

0.0001 0.3165 0.7890 0.4303 0.0419 0.1182 0.3322 0.0368 0.7247 0.6333 0.7770 0.8303 0.0001 0.0042 0.1585 0.6646 0.69

The highest content of phenolic compounds is usually obtained after extraction with aqueous. Due to toxicity of methanol for this research as extraction solvent we have used aqueous ethanol. According to our results, in gentian root extracts TPC varied between 18.0 and 38.3 mg GAE/ g dw. Azman et al. (2014) reported different value for TPC in 50% methanolic extract (12.03 mg GAE/g dw), which could be the consequence of geographical or ecological variations and time of harvest. Our data showed that the highest TPC was achieved after employment of the subsequent parameters: X1: 35 min, X2: 50% EtOH, X3: 1:30 and X4: 50 °C. At the same time, lowest TPC was obtained employing parameters: X1: 35 min, X2: 90% EtOH, X3: 1:30 and X4: 50 °C. Results of ANOVA showed that among tested exctraction parameters, SS ratio (X3) showed major signifficant effect on TPC followed by ethanol concentration (X2) (Fig. 1). On the Fig. 2, the impact of extraction parameteres on TPC was presented using graphs formed by settling two independent variables at values of central design and changing the remaining ones. The increase in SS ratio was positively associated with TPC increase. This result is a consequence of the mass transfer postulates. According to them gradient difference between the solid and solvent concentrations is seen as mass transfer initiator (Belwal et al., 2016). On the other hand, lower ethanol concentrations means higher content of total polyphenols in extract. It is well known that water is a good extraction solvent for polyphenolic compounds. Still, in many cases TPC is lower after water extraction compared to extraction with aqueous ethanol. Polyphenols in plant matrix are usually bound to proteins and/or polysaccharides by hydrogen and hydrophobic bounds. Therefore, besides high solvency, it is important for solvent to have ability for cleavage of these bonds (Miralai et al., 2008). That is the reason why binary sistems are usually more functional in the extraction of polyphenols in comparison with mono-solvent systems (Chew et al.,

* p values lower than 0.05 are statistically significant.

experimental conditions are given in Table 2. These results were adjusted to quadratic polynomial model and ANOVA test was used for estimation of effects of analysed variables, their interactions, and statistical significance of the model. Results of ANOVA analysis are presented in Table 3. According to p values that were statistically significant quadratic polynomial model indicated good estimation for tested responses. Satisfactorily high coefficients of multiple determinations (R2) with values 0.87, 0.98 and 0.69 for TPC, isogentisin, and gentiopicroside content, respectivelly, also confirmed this result. Insignificant lack of fit (p > 0.05) showed the good efficency of the models as well. 3

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Mustafa et al., 2015b), but this could be the consequence of different solvent used for the extraction. Although methanol was more effective, aqueous ethanol is preferred solvent in food industry. The highest gentiopicroside content was gained through employment of the following conditions: X1: 35 min, X2: 50% EtOH, X3: 1:30 and X4: 50 °C. In contrast, the lowest gentiopicroside content was reached by means of next conditions: X1: 35 min, X2: 90% EtOH, X3: 1:30 and X4: 80 °C. Results of ANOVA demonstrated that among tested exctraction parameters, extraction temperature (X4) had signifficant and positive influence on gentiopicroside content (Figs. 1 and 2). This is in line with previous data reported by Suomi et al. (2000). They reported that content of two iridoid glucosides (aucubin and catalpol) extracted from Veronica longifolia raised with increase in extraction temperature. Lee et al. (2012) exhibited that optimal temperature for extraction of three iridoid glucosides (morronoside, loganin and cornoside) from the orni fruit (Cornus officinalis) is 69.8 °C. The variables that also affected gentiopicroside content in our investigation were quadratic terms of EtOH concentration and SS ratio. Other factors, their quadratic terms as well as interactions obtained between them were non-significant (p < 0.05). The capacity for extraction of maximal gentiopicroside amount from gentian root can be explained with definite predictive equation, taking into consideration only significant parameters: Gentiopicroside content (mg/g dw) = 12.9175 + 0.041875X1 + 0.160484X2 + 0.219563X3 + 0.018X4 – 0.0008375X1X2 – 0.00131172X22 – 0.00365938X32 According to our results maximal gentiopicroside content can be extracted after application of the following parameters: extraction time – 64 min, ethanol concentration – 41%, SS ratio – 1:29 and extraction temperature - 80 °C. Similarly to our results, Nastasijević et al. (2016) showed that among four type of gentian root extracts, the one obtained with 50% EtOH yield the highest abundance of gentiopicroside. Fig. 1. Pareto charts of significant influence of factors, their interactions, as well as quadratic effects on the TPC (a), gentiopicroside content (b) and isogentisine content (c). Factor codes: A – extraction time, B – ethanol concentration, C – SS ratio, D – extraction temperature; AB, BD, CD – their interactions; AA, BB, CC, DD – quadratic effects.

3.4. Influence of extration variables on isogentisin content Isogentisin content in gentian root extracts achieved using UAE technique differed from 2.48 to 4.93 mg/g dw. The obtained results were higher as compared to values formerly published in the literature (Aberham et al., 2011; Citova et al., 2008). In line with our data the maximal isogentisin content after UAE was reached by application of the following conditions: X1: 35 min, X2: 50%, X3: 1:30 and X4: 80 °C. At the same time, the minimal isogentisin content was obtained after application of following parameters: X1: 35 min, X2: 10%, X3:1:30 and X4: 50 °C. ANOVA showed that the extraction of isogentisin was under significant and positive influence of temperature (X4), ethanol concentration (X2), SS ratio (X3) and time of extraction (X1) (in declaining order) (Figs. 1 and 2). The quadratic levels of SS ratio, EtOH concentration and temperature also significantlly influenced isogentisin content. Considering interactions among variables, only interaction among ethanol concentration and temperature showed significant negative effect on the TPC. After eliminating non-significant factors the final predicitve equation for describing the efficancy of isogentisin extraction from gentian root is as followes:

2011). Gentian root is a good source of polysaccharides (inulin and pectin) which can serve as polyphenols binders. According to our results quadratic effects of SS ratio and ethanol concentration significantly influenced TPC. Remaining parameters and interactions between them were insignificant. After removing non-significant factors the final prognostic equation for characterizing the capacity for achieving the maximum TPC in extracts is as stated below: TPC (mg GAE/g dw) = 6.52897 + 0.759727X2 + 0.63901X3 – 0.19625X4 – 0.00829102X22 - 0,0115391X32 + 0.00654167X3X4 After carrying out detailed experimental design we determined that optimal parameters for TPC extraction are extraction solvent 53% EtOH, SS ration 1:49, time of extraction 42 min and temperature of 80 °C.

Isogentisin content (mg/g dw) = -1,03786 + 0,0135972X1 + 0,131342X2 + 0,0504881X3 + 0,0104603X4 - 0,0001875X1X2 0,000966964X22 - 0,000416667X2X4 - 0,000655357X32 + 0,00025873X42

3.3. Influence of extraction variables on gentiopicroside content The most abundant secoiridoid compound in gentain root is gentiopicroside, and its concentration in extract depends on applied extraction procedure. According to our results gentiopicroside content in gentian root extracts achieved with UAE technique differed from 18.47 to 22.92 mg/g dw. These values are slightly lower compared to previously published data (Carnat et al., 2005; Aberham et al., 2011;

Results achieved in our investigation demonstrated following parameters as optimal for extraction of maximal isogentisin content: extraction time – 65 min, EtOH concentration – 44%, SS ratio – 1:38 and extraction temperature - 80 °C. Nastasijević et al. (2016) tested biological activity of four types of 4

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Fig. 2. Response surface plots for the effect of (a) time/ethanol concentration and (b) SS ratio/temperature on TPC (I), gentiopicroside content (II) and isogentisin content (III).

gentian root extracts: water extract, 25%, 50% and 75% ethanol extracts. Chemical analysis showed that isogentisin was present only in 50% and 75% ethanol extracts. Similar to our results, Joubert et al. (2012) indicated that the maximal recovery of xanthones from Cyclopia subternata was obtained using ethanol-water (1:1) mixture. Also, Bosman et al. (2017) recorded that the highest concentration of xanthone compounds was extracted from Cyclopia genistoides with ethanol concentrations between 20% and 60% EtOH.

Table 4 Comparison between predicted and experimentally obtained values for investigated responses. Response values (mg/g dw)

Predicted value

Experimental value

TPC Gentiopicroside content Isogentisin content

37.94 22.00 4.93

36.55 ± 0.31 21.45 ± 0.22 4.81 ± 0.09

dw – dry weight; TPC - total polyphenols content.

3.5. Determination and experimental verification of obtained optimal conditions

4. Conclusion In this study, we used RSM for the identification of significant variables and their intearactions affecting extraction of bioactive compounds from G. lutea root. According to our results temperature, SS ratio and solvent concentration were essential parameters affecting the content of isogentisin, gentiopicroside and TPC in G. lutea extracts. Predicted values developed using RSM were confirmed by the experimental values. Obtained results create a base for further research such as bioactivity assays and purification of selected phenolic compounds.

After employing the desirability function method for all of the analysed responses (TPC as well as amounts of individual compounds isogentisin and gentiopicroside) it can be determined that optimal parameters for the UAE of their maximal content from gentian root are in the following manner: X1: 31 min, X2: 49%, X3: 1:42, and X4: 80 °C. Projected values of responses achieved under optimal conditions using calculated models were determined and validated experimentally employing the same UAE procedure. Results acquired through validation of optimized conditions were near to predicted values (Table 4). This supports application of chosen RSM model for UAE of gentian root for the purpose of maximizing TPC, and content of two biologically active compounds (isogentisin and gentiopicroside).

Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, project number 5

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