Optimisation of polyphenol extraction from Hypericum

Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 1–8

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Optimisation of polyphenol extraction from Hypericum perforatum (St. John’s Wort) using aqueous glycerol and response surface methodology Blagoj Karakashov a , Spyros Grigorakis a , Sofia Loupassaki a , Dimitris P. Makris b,∗ a Food Quality & Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (M.A.I.Ch.), Centre International de Hautes Etudes Agronomiques Méditerranéennes, P.O. Box 85, Chania 73100, Greece b School of Environment, University of the Aegean, Mitr. Ioakim Street, Myrina 81400, Lemnos, Greece

a r t i c l e

i n f o

Article history: Received 2 August 2014 Accepted 30 November 2014 Available online 19 March 2015 Keywords: Extraction Glycerol Hypericum perforatum Polyphenols

a b s t r a c t Hypericum perforatum is a plant rich in biologically functional polyphenolic phytochemicals. The efficient recovery of polyphenols from this material has not been previously investigated in depth, in terms of establishing an environmentally benign and cost effective process that would aim at producing extracts enriched in polyphenolic compounds. The objective of the present study was the examination of the efficiency of water/glycerol mixtures in extracting polyphenols from dried aerial parts of H. perforatum and its critical comparison with water. Extraction temperature and time were optimised using response surface methodology, while comparative assessment of the extraction efficiency between water/glycerol and water was carried out using kinetics. The results showed that 10% (w/v) aqueous glycerol at 70 ◦ C provided very satisfactory extraction yield in total polyphenols (89.9 mg GAE g−1 dw), which was significantly higher than that attained with water. Similar results were drawn for the reducing power, in concurrence with the polyphenolic concentration. Liquid chromatography–mass spectrometry analysis showed that the polyphenolic profile of water/glycerol extract was composed by polar substances, such as chlorogenic acids and quercetin glycosides. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction The objectives concerning the exploitation of medicinal plants include the recovery of fine chemicals and production of precious metabolites, such as natural antioxidants,

Abbreviations: AAE, ascorbic acid equivalents; CCD, central composite design; EtOH, ethanol; GAE, gallic acid equivalents; LC-DAD–MS, liquid chromatography-diode array–mass spectrometry; TP, total polyphenols; TPTZ, 2,4,6-tripyridyl-s-triazine. ∗ Corresponding author. Tel.: +30 22540 83114. E-mail address: [email protected] (D.P. Makris). http://dx.doi.org/10.1016/j.jarmap.2014.11.002 2214-7861/© 2015 Elsevier GmbH. All rights reserved.

which are value-added products of high significance to the pharmaceutical, cosmetics and food industries (Galanakis, 2012). The search for plants as cost-effective sources of multifunctional phytochemicals embraces strategies in the direction of (i) exploiting materials with high content and rich composition, (ii) deploying effective recovery processes and (iii) ascertaining production of novel formulations without further generation of waste. In this line, the use of low-cost, non-toxic solvent systems for the recovery of target compounds becomes imminent. This is a highly significant concept, since the value of a wide spectrum of similar studies pertaining to solid–liquid extraction of bioactive phytochemicals would not go

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Nomenclature Cgl De dw h k kB PR rs t tR T YTP YTP(s)

glycerol concentration (%, w/v) diffusion (m2 s−1 ) dry weight (g) initial extraction rate (mg g−1 min−1 ) extraction rate constant (g mg−1 min−1 ) Boltzmann constant (J K−1 ) reducing power (mM AAE g−1 dw) effective particle radius (m) time (min) time required to reach regular regime (min) temperature (◦ C) extraction yield in total polyphenols (mg GAE g−1 dw) extraction yield in total polyphenols at saturation (mg GAE g−1 dw)

Greek letters dielectric constant (dimensionless) ε  viscosity (kg/(s m))  dynamic viscosity (mPa s)

beyond laboratory-scale level. This is because the solvents tested to achieve high recovery yields are toxic and/or highly flammable (methanol, acetone, ethyl acetate), or expensive due to restrictions arising from state laws (ethanol) and therefore completely incompatible with a prospect industrial, “green” extraction process. Removal of these solvents from extracts destined for food, cosmetic or pharmaceutical formulations would entail strict quality control, recycling and appropriate safe handling, with an increased associated cost. The genus Hypericum is widely distributed through the temperate regions and is represented by approximately 450 species. Among the Hypericum genus, the therapeutic use and the commercial value of Hypericum perforatum, also known as St. John’s Wort, are notorious, as various preparations from this plant have been used for decades to treat a spectrum of ailments, owed to its favourable combination of high content in various bioactive metabolites, wide geographical occurrence and long ethnopharmacologic history. H. perforatum has to-date gained acceptance as a mild-to-moderate anti-depressant, as well as a remedy to a wide variety of conditions (Hammer and Birt, 2014). H. perforatum extracts contain many different classes of constituents including flavonoids and biflavonoids, phloroglucinols, naphthodianthrones, caffeic acid derivatives, etc., many of which may be responsible for the anti-inflammatory activity, in line with anti-depressant data. Though a plant of high pharmacological potency, data on the optimisation of the extraction of bioactive components from H. perforatum are very scarce, while the studies pertaining to this issue suffer serious shortcomings, such as the toxicity of the solvents used (Aybastıer et al., 2013). Glycerol is a natural, non-toxic and low-cost substance and recent reports indicated that it can favourably change the polarity of water, acting as an efficient co-solvent for

increased polyphenol recovery (Apostolakis et al., 2014). In this frame, the scope of this study was a thorough investigation on the potential of water/glycerol media for polyphenol extraction from the aerial parts of H. perforatum, employing a carefully designed process, based on response surface methodology and kinetics. 2. Materials and methods 2.1. Chemicals and reagents All solvents used for chromatographic analyses were HPLC grade. Ferric chloride hexahydrate was from Acros Organics (New Jersey, USA). Gallic acid, ascorbic acid, Folin–Ciocalteu reagent and 2,4,6-tripyridyl-s-triazine (TPTZ) were from Sigma–Aldrich (Steinheim, Germany). Glycerol was from Fisher Scientific (New Jersey, USA). 2.2. Plant material The plant material used in this study consisted of the aerial parts of H. perforatum L., which was provided by the M.A.I.Ch. Herbarium, where a voucher specimen was deposited (Herb. no. 9223). After collection, the plant tissue was left to dry at room temperature in a dry and dark chamber for 7 days, and then ground to a fine powder using a domestic blender (Bosch MMB 112R, Stuttgart, Germany). This material was used for all further procedures. 2.3. Extraction procedure An amount of approximately 1 g of the dry, ground material was placed in a 100-mL glass vial with 50 mL of extracting medium, composed of either deionised water or 10% (w/v) aqueous glycerol. The glycerol level was chosen on the basis of a previous examination (Apostolakis et al., 2014) and preliminary experimentation. Extractions were carried out under stirring with a teflon-coated magnetic stirrer, at 400 rpm, for predetermined time periods. During extractions, the glass vial was immersed in an oil-bath, heated by a hot plate (YellowLine MST Basic C, Richmond, VA), set at 50, 60 and 70 ± 1.5 ◦ C. Temperature was controlled by a YellowLine TC1 thermostat. Sampling was accomplished at regular intervals (5–95 min) and the extracts obtained were filtered through paper filter and stored at −20 ◦ C until analysed. All extracts were also filtered through 0.45-␮m syringe filters prior to determinations. 2.4. Determination of total polyphenol yield (YTP ) In a 1.5-mL Eppendorf tube, 0.78 mL of distilled water, 0.02 mL of sample appropriately diluted, and 0.05 mL of Folin–Ciocalteu reagent were added and vortexed. After exactly 1 min, 0.15 mL of aqueous sodium carbonate 20% was added, and the mixture was vortexed and allowed to stand at room temperature in the dark, for 60 min. The absorbance was read at 750 nm in a HP 8452A diodearray spectrophotometer (Santa Clara, CA) and the total polyphenol concentration was calculated from a calibration curve, using gallic acid as a standard. Results were

B. Karakashov et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 1–8 Table 1 Experimental values and coded levels of the independent variables used for the central composite experimental design. Independent variables

Code units

t (min) T (◦ C)

X1 X2

0

1

5 50

50 60

95 70

expressed as mg gallic acid equivalents (GAE) per gram of dry weight. Total polyphenol yield (YTP ) was calculated as follows (Makris and Kefalas, 2013): YTP (mg GAE g−1 dw) =

C ×V m

(1)

where C is the TP concentration of the extract (mg L−1 ), V is the volume of the extraction medium (L) and m is the dry weight (g) of the plant material. 2.5. Determination of the reducing power (PR ) The assay measures the ability of antioxidants to reduce the ferric TPTZ-complex [Fe(III)-TPTZ]3+ to the intensely blue coloured ferrous complex [Fe(II)-TPTZ]2+ in acidic medium. PR values are calculated by measuring the absorbance at 620 nm and relating it to a standard solution of ascorbic acid, a natural, strong reducing agent. As this assay measures the reducing capacity based upon reduction of ferric ion, it is specific to polyphenolic antioxidants. Determinations were performed according to a previously established protocol (Makris et al., 2007). Sample (0.05 mL) appropriately diluted with methanol was mixed thoroughly with 0.05 mL FeCl3 solution (4.03 mM in 0.05 M HCl), and incubated for 30 min in a water bath at 37 ◦ C. Following this, 0.9 mL TPTZ solution (1 mM in 0.05 M HCl) was added, and the absorbance was recorded at 620 nm after exactly 5 min. PR was determined as mM ascorbic acid equivalents (mM AAE) per g of dw. 2.6. Experimental design and response surface methodology Optimisation of the extraction process with respect to temperature (T) and resident time (t) was achieved by implementing a central composite design (CCD). The two independent variables (T and t) were coded at three levels, −1, 0 and 1 (Table 1), according to the following equation: xi =

Xi − X0 , Xi

xi = 1, 2

Table 2 Measured and predicted YTP determined for individual design points. Design point

Independent variables

Responses YTP (mg GAE g−1 dw)

X1

Measured

Predicted

1 2 3 4 5 6 7 8 9 10

−1 (5) −1 (5) 1 (95) 1 (95) −1 (5) 1 (95) 0 (50) 0 (50) 0 (50) 0 (50)

50.15 68.44 59.42 84.73 58.10 60.46 59.86 86.18 64.42 67.61

50.99 70.79 56.79 83.60 54.91 64.22 61.65 84.96 67.33 67.33

Coded variable level −1

(2)

where xi and Xi are the dimensionless and the actual value of the independent variable i, X0 is the actual value of the independent variable i at the central point, and Xi is the step change of Xi corresponding to a unit variation of the dimensionless value. Response (YTP ) at each design point was recorded (Table 2). The data obtained were subjected to regression analysis using least square methodology, to extract the equation that provided the response values as a function of the independent variables (mathematical model). Analysis of variance (ANOVA) was used to assess the statistical significance of the model. The significance was improved through

3

X2 −1 (50) 1 (70) −1 (50) 1 (70) 0 (60) 0 (60) −1 (50) 1 (70) 0 (60) 0 (60)

a “backward elimination” process, by omitting the insignificant dependent terms (p > 0.05). Response surface plot was obtained using the fitted model, by maintaining the independent variables simultaneous. 2.7. Liquid chromatography-diode array–mass spectrometry A modified method previously reported (Kalogeropoulos et al., 2010) was used. A Finnigan MAT Spectra System P4000 pump was used coupled with a UV6000LP diode array detector and a Finnigan AQA mass spectrometer (Waltham, MA). Analyses were carried out on an endcapped Superspher RP-18, 125 mm × 2 mm, 4 ␮m, column (Merck, Germany), protected by a guard column packed with the same material, and maintained at 40 ◦ C. Analyses were carried out employing electrospray ionisation (ESI) at the positive ion mode, with acquisition set at 10 and 70 eV, capillary voltage 4 kV, source voltage 25 V, detector voltage 650 V and probe temperature 400 ◦ C. Eluent (A) and eluent (B) were 2.5% acetic acid and methanol, respectively. The flow rate was 0.33 mL min−1 , and the elution programme used was as follows: 0–2 min, 0% B; 2–20, 60% B; 40, 40% B, 60, 40% B. 2.8. Kinetics and statistical analysis All determinations were carried out at least in triplicate and values were averaged. Response surface experimental design and statistics were performed with JMPTM 10. Kinetics was established by non-linear regression between YTP and t. Non-linear regressions were performed with SigmaPlotTM 12.0, at least at a 95% significance level. 3. Results and discussion 3.1. Response surface optimisation The independent variables (X1 and X2 ), as well as measured and predicted values for the response (YTP ) are analytically presented in Table 2. The values of YTP obtained experimentally were analysed by multiple regression to fit a 2nd-order polynomial equation. After removal of the

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B. Karakashov et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 1–8 100

YTP (mg GAE g-1 dw)

80

60

40

Extraction with 10% (w/v) glycerol Extraction with water Fitted model

20

0 0

20

40

60

80

100

120

t (min)

Fig. 2. Non-linear regression between YTP and t values, for the extraction of TP from H. perforatum. Extractions were carried out under stirring at 400 rpm, 70 ◦ C and a liquid-to-solid ratio of 50 mL g−1 .

Fig. 1. Response surface plot showing variation of YTP as a function of simultaneous changes in T and t. Extractions were carried out with 10% (w/v) aqueous glycerol, at a liquid-to-solid ratio of 50 mL g−1 .

The general mathematical expression that relates viscosity (), diffusion (D) and temperature (T) is the Stokes–Einstein equation: D=

non-significant factors, the theoretical model would be: YTP (mg GAE g−1 dw) = 1.81 − 0.004t 2 + 0.102t + 1.165T (3) The significance of fitting was checked using the square coefficient of correlation (R2 ), which was 0.96 (p = 0.0056). This outcome suggested a satisfactory agreement between observed and predicted responses and that Eq. (3) can efficiently predict the experimental results. The deployment of the model allowed for the theoretical calculation of the optimal set of conditions, that were T = 70 ◦ C and t = 68.7 min. Under these conditions, the optimal theoretical YTP was calculated 86.28 ± 6.54 mg GAE g−1 dw. The trend revealed was recorded in the form of a threedimensional plot (Fig. 1).

3.2. Extraction kinetics The extraction of various components from plant material implicates diffusion phenomena (Bogdanov and Svinyarov, 2013; Herodeˇz et al., 2003; RakotondramasyRabesiaka et al., 2010; Sant’Anna et al., 2013; Seikova et al., 2004) and generally includes three phases: (i) penetration of solvent into the mass of the solid particles and solubilisation of the compounds in question (fast, non-rate limiting); (ii) diffusion of the solubilised compounds from the inner particle to the solid/liquid interface, termed as internal diffusion (slow, rate-limiting); and (iii) diffusion of the solubilised compounds from the solid/liquid interface into the solution, termed as external diffusion (generally considered as non-rate limiting) (Aguilera, 2003). Diffusion process occurs due to concentration gradient, according to Fick’s law.

kB T 6rs

(4)

where kB is the Boltzmann’s constant and rs is the effective radius of the diffusing molecule. Eq. (4) dictates that the higher the , the lower the D. Glycerol is a viscous liquid having a dynamic viscosity () equal to 1400 mPa s, at 20 ◦ C (Elson, 2007); therefore it would be reasonably anticipated that mixing glycerol with water ( = 1.002 mPa s) would raise the mixtures viscosity, thus hindering diffusion, unless increased temperature is applied to compensate for the higher viscosity. The dynamic viscosity  of a mixture of two liquids A and B is given by the following equation (Naidu and Krishnan, 1966): =

1 (XA /A ) + (XB /B )

(5)

where XA and XB are the mole fractions, and A and B are the pure components’ viscosities for component A and B, respectively. Using Eq. (5), and taking into consideration the aforementioned  values for water and glycerol, it can be calculated that a 10% (w/v) aqueous solution of glycerol would have  = 1.023 mPa s, which is approximately by 2% higher than that of water. This very low difference in viscosity would not change De significantly, and extraction yield would not be affected to a measurable extent. On the other hand, glycerol has a dielectric constant ε = 46.5 (Lameiras et al., 2011), which could lower that of water (ε = 80.1), thus making it less polar and more suitable for efficient polyphenol extraction (Cacace and Mazza, 2002). With the view to verifying the above hypothesis, a comparative examination was carried out, based on kinetics. Kinetics was established by performing non-linear regression between YTP and t, for extractions carried out by both water and 10% (w/v) glycerol, at 70 ◦ C, to enable the determination of kinetic parameters that were descriptive of the extraction process.

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Table 3 Parameters of 2nd-order kinetics, determined for the extraction of TP from H. perforatum, at 70 ◦ C, using the two different extraction media. Kinetic parameters

10% glycerol Water

k (min g mg−1 ) × 10−3

h (mg g−1 min−1 )

tR (min)

YTP(s) (mg GAE g−1 dw)

8.12 14.41

65.63 86.58

1.37 0.89

89.90 77.51

1.4

Extraction with 10% (w/v) glycerol Extraction with water

1.0

-1

t/YTP (min g mg )

1.2

0.8

0.6

0.4

0.2

0.0 0

20

40

60

80

100

120

t (min)

Fig. 3. Second-order kinetics of TP extraction from H. perforatum. Extractions were carried out under stirring at 400 rpm, 70 ◦ C and a liquid-to-solid ratio of 50 mL g−1 .

The curve fitted to the experimental data (Fig. 2) described 2nd-order extraction kinetics, considering the boundary conditions t = 0 to t and YTP(t) = 0 to YTP(t) (Ho et al., 2005; Rakotondramasy-Rabesiaka et al., 2007): YTP(t) =

2 YTP(s) kt

1 + YTP(s) kt

(6)

where YTP(s) and k represent the TP yield at saturation and the extraction rate constant, respectively. Fitting for both 10% (w/v) aqueous glycerol and water, and for every temperature tested, was statistically significant (R2 > 0.99, p < 0.0001), suggesting that YTP can be reliably predicted as a function of t, using Eq. (6). Considering the concessions that (i) polyphenols were extracted from the solid particles into the solution through diffusion, and (ii) at saturation conditions YTP remained constant, the 2nd-order extraction kinetics is illustrative of two extractions phases: first, diffusion was fast, presumably due to fast solubilisation of the extracting compounds and the high concentration gradient; second, diffusion was paced down because of the lower concentration gradient and to longer time required for the extraction of the less easily extracted compounds. Transformation of Eq. (6) provides a linearised form: t 1 t = + 2 YTP(t) YTP(s) kYTP(s)

(7)

when t approaches 0, the initial extraction rate, h, given as YTP(t) /t, is defined as: 2 h = kYTP(s)

(8)

If t/YTP(t) is plotted versus t, a straight line expressed by the equation y = ax + b is obtained (Fig. 3), where a = 1/YTP(s) and

b = 1/h. Hence the kinetic parameters YTP(s) , k and h could be determined graphically. For both extraction media, the correlations between t/YTP(t) and t were very high and statistically significant (R2 > 0.999, p < 0.0001), which permitted the credible estimation of the kinetic parameters (Table 3). Moreover, it has been proposed that the change in the kinetic trend, from fast to slow, occurs when about 80% of the extraction efficiency has been attained (Seikova et al., 2004). This period, called “regular regime” characterises the second, slow phase of the extraction, where small increases in the extraction rate occur within substantial amount of time. The time required for the extraction process to reach the regular regime, tR , was proposed to be estimated by the following equation (RakotondramasyRabesiaka et al., 2010): tR =

1 kYTP(s)

(9)

The tR values determined for the cases examined, using Eq. (9), are also given in Table 3. The kinetic parameters k, h and tR were higher when the extraction was performed with water, which indicated a faster extraction rate. By contrast, the YTP(s) was 13.8% higher in extracts obtained with 10% (w/v) glycerol. Considering that internal diffusion is the rate-limiting step, it might be assumed that polyphenol diffusion within the solid particles was faster with water. However, the higher YTP(s) value achieved with 10% (w/v) glycerol was a clear evidence that aqueous glycerol acted as a more efficient extracting medium, presumably because of its lower ε. Therefore, the extraction process with the water/glycerol system, though slower, provided higher YTP . 3.3. Extraction yield and phytochemical profiling The highest YTP (89.9 mg GAE g−1 dw) was achieved after 95 min of extraction, at 70 ◦ C. According to the bibliographic data on the total polyphenol content of H. perforatum extracts, obtained using various solvent systems and techniques (Table 4), the values reported vary within a very large range, from 9.13 to 461.19 mg GAE g−1 dw, with an average of approximately 135 mg GAE g−1 dw. However, these data should be interpreted with caution, because the amount of polyphenols in H. perforatum tissues depends on a number of variables involved, such as intraspecific chemodiversity, plant breeding, developmental stage, post-harvest handling, biotic and abiotic factors (Bruni and Sacchetti, 2009). Furthermore, variations in composition may also be related to environmental conditions that may differ largely from region to region or from the genetic diversification that may occur among regionally distinct subspecies (Boˇzin et al., 2013).

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Table 4 Bibliographic data on the efficiency of various methodologies with regard to polyphenol recovery from H. perforatum. Extraction method

Yield (mg GAE g−1 dw)

Reference

10% (w/v) aqueous glycerol, 70 ◦ C, 95 min Ethanol, Soxhlet Methanol, maceration for 8 h Methanol/HCl, ultrasonication Ethanol/water (7:3), maceration 72 h Ethanol/water (5:5), maceration 7 days Methanol and ethanol/water (6:4) Methanol, maceration 72 h Boiling water, 5 min Methanol/acetone (1:1), Soxhlet Methanol

86.60 71.6 355.01 461.19 15.54 25 191 15.01 143.2 9.13 64.08

This method Ali et al. (2011) Altun et al. (2013) Aybastıer et al. (2013) Boˇzin et al. (2013) Danila et al. (2011) Gioti et al. (2009) Kalogeropoulos et al. (2010) Rainha et al. (2011) Sagratini et al. (2008) Zugic´ et al. (2014)

Another matter pertaining to the differences in the levels of polyphenols found in various studies is the extraction medium. In most cases reported in the literature, extraction was carried out using solvents such as methanol and acetone, which are toxic and not compatible with industrial applications for food, cosmetics or pharmaceutical manufacturing. Moreover, the use of similar solvent systems on an industrial scale would raise issues of solvent handling and disposal, thus making the production process costly and environmentally invidious. By contrast, this study was carried out with the objective of developing an environmentally benign extraction process, using nontoxic extracting media. At this point, it should be noted that polyphenols may be easily solubilised in polar protic media, such as hydroethanolic and presumably, hydroglycerolic mixtures, while different fractions can be obtained through modification of the polarity of the medium, by varying ethanol (or glycerol) concentration (Galanakis et al., 2013; Tsakona et al., 2012). The analysis of the richest extract obtained by LC-DAD–MS revealed the presence of 13 major phytochemicals (Table 5). Based on previous information (Orˇcic´ et al., 2011; Silva et al., 2005; Tatsis et al., 2007), these components were tentatively identified as hydroxycinnamate derivatives (peaks 1–6), flavanols (peak 7) and flavonol glycosides (8–13). This finding is consistent with studies that reported the major polyphenolic phytochemicals in H. perforatum to be caffeoyl- and p-coumaroyl-quinic acid conjugates and quercetin glycosides (Hosni et al., 2010; Kalogeropoulos et al., 2010; Silva et al., 2005; Tatsis

et al., 2007). However, regarding other metabolites commonly occurring in H. perforatum, such as hypericins and hyperforins, only hypericin was detected. Hypericins and hyperforins are substances possessing peculiar bioactivities, which lend H. perforatum its anti-depressant properties, hence they are considered integral to the quality of H. perforatum preparations (Caccia, 2006). These substances are significantly less polar compared with either caffeates or quercetin glycoconjugates and their solubility in water is very low (Jürgenliemk and Nahrstedt, 2003); thus it is quite probable that aqueous glycerol acted selectively, extracting only the most polar substances. Indeed, the evidence emerged from a recent study on the extraction of polyphenols from olive leaves using aqueous glycerol (Apostolakis et al., 2014), suggested this extracting medium to be selective for polar phenolics. This is probably the reason for the absence of other hypericins and hyperforins from the extract analysed. This effect needs further investigation, since an extracting medium composed of varying water/glycerol amounts could be used as a means of obtaining H. perforatum fractions enriched in specific phytochemicals, thus enabling the formulation of products with more targeted bioactivities. On the other hand, the effect of temperature should not be disregarded. The temperature employed (70 ◦ C) might be prohibited for the extraction of these components, but since there are no studies on the thermostability of hypericins and hyperforins, no credible claims could be made on this issue. However, it is to be emphasised that previous studies reported that anthocyanin extraction at

Table 5 UV–vis and mass spectral characteristics of the main phytochemicals detected in the H. perforatum extract at 70 ◦ C, after 95 min. Peak #

Rt (min)

UV–vis

[M+H]+ (m/z)

Other ions (m/z)

Tentative identity

1 2 3 4 5 6 7 8 9 10 11 12 13 14

7.45 7.76 9.74 10.13 12.04 12.31 14.04 20.87 21.25 21.61 23.12 23.49 23.91 60.00

328 320 308 310 320 318 280 256, 356 256, 356 256, 360 252, 358 264, 352 266, 328 274

355 355 339 339 579 579 291 465 611 611 449 449 595 505

163 377 (Na+ 361 (Na+ 361 (Na+ 601 (Na+ 601 (Na+ 313 (Na+ 487 (Na+ 633 (Na+ 633 (Na+ 471, 303 471, 303 617 (Na+ 483

Caffeoylquinic acid Caffeoylquinic acid p-Coumaroylquinic acid p-Coumaroylquinic acid Chlorogenic acid derivative Chlorogenic acid derivative Catechin Quercetin 3-O-galactoside Quercetin rutinoside Quercetin rutinoside Quercetin rhamnoside Quercetin rhamnoside Quercetin rhamnoside derivative Hypericin

adduct), 163 adduct), 147 adduct), 147 adduct), 355 adduct), 355, 163 adduct) adduct), 303 adduct), 465, 303 adduct), 465, 303

adduct), 449, 287

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and/or antagonism, which depend on the relative amounts of the compounds interacting (Karvela et al., 2013). 4. Conclusions

Solvent

W/Gl

W

0

1

2

3

4

5

6

PR (mM AAE g-1 dw)

Fig. 4. Comparative diagram displaying PR values of the H. perforatum extracts, obtained with water and 10% (w/v) aqueous glycerol, at 70 ◦ C after 95 min. Assignments: W/Gl, 10% (w/v) aqueous glycerol; W, deionised water.

temperatures above 35 ◦ C gave reduced extraction yields (Cacace and Mazza, 2003). Likewise, total polyphenol extraction from onion solid wastes was negatively affected by temperatures higher than 40 ◦ C (Khiari and Makris, 2009).

3.4. Reducing power All the classes of polyphenolic substances found in the H. perforatum extract, have been demonstrated to exhibit appreciable antioxidant properties, the most potent being quercetin glycosides and the aglycone (Silva et al., 2008), while other metabolites such as hypericins, display notably lower efficacy (Orˇcic´ et al., 2011). PR is credible criterion for the evaluation of the antioxidant activity, as reported in studies on various polyphenol-containing materials (Makris et al., 2007), where a statistically significant correlation between the amount of polyphenols and PR was demonstrated. Dose-depended reducing effects have been demonstrated for H. perforatum extracts and concurred with other antioxidant properties (Altun et al., 2013). The determination of PR in the extracts obtained with both 10% (w/v) glycerol and water after 95 min, at 70 ◦ C, showed that aqueous glycerol extracts exhibited 9% higher potency (Fig. 4). This was in line with the results on the YTP and consisted clear evidence that differences in YTP were reflected on the antioxidant properties of the extracts. This assumption is in accordance with previous examinations on various Hypericum species, where it was shown that extracts with the higher polyphenolic load also exhibited stronger antioxidant effects (Boˇzin et al., 2013; Rainha et al., 2011). Similar results were reported for several Hypericum taxa (Sagratini et al., 2008). Therefore, the differences in the PR values between the two extracts are rather attributed to differences in the total polyphenol concentration and the relative amounts of the various polyphenols. The latter becomes important because the expression of the antioxidant activity might embrace phenomena of synergism

Glycerol is a natural, non-toxic and inexpensive substance, which has significantly lower dielectric constant compared with water, and thus it could improve water’s capacity to extract polyphenolic compounds from solid material. This hypothesis was substantiated with a detailed kinetic study on extraction of polyphenols from H. perforatum, which showed (i) that incorporation of glycerol at a level of 10% (w/v) into water does not affect its viscosity and (ii) that glycerol-containing aqueous solutions are more potent in retrieving polyphenols, at 70 ◦ C, compared with water. The higher recovery of polyphenols was found to be reflected on the ferric-reducing ability of the extracts obtained. Liquid-chromatography–mass spectrometry analyses disclosed that the polyphenolic profile of the extracts, obtained either with aqueous glycerol or water, is dominated by chlorogenic acids and quercetin glycosides, which are the principal antioxidant components. References Aguilera JM, 2003. Solid–liquid extraction. In: Tzia C, Liadakis G (Eds.), Extraction Optimization in Food Engineering. Marcel Dekker Inc., Switzerland. Ali M, Arfan M, Ahmad H, Zaman K, Khan F, Amarowicz R, 2011. Comparative antioxidant and antimicrobial activities of phenolic compounds extracted from five Hypericum species. Food Technology and Biotechnology 49, 205–213. Altun ML, Yılmaz BS, Orhan IE, Citoglu GS, 2013. Assessment of cholinesterase and tyrosinase inhibitory and antioxidant effects of Hypericum perforatum L. (St. John’s Wort). Industrial Crops and Products 43, 87–92. Apostolakis A, Grigorakis S, Makris DP, 2014. Optimisation and comparative kinetics study of polyphenol extraction from olive leaves (Olea europaea) using heated water/glycerol mixtures. Separation and Purification Technology 128, 89–95. Aybastıer O, S¸ahin S, Demir C, 2013. Response surface optimized ultrasonic-assisted extraction of quercetin and isolation of phenolic compounds from Hypericum perforatum L. by column chromatography. Separation Science and Technology 48, 1665–1674. Bogdanov MG, Svinyarov I, 2013. Ionic liquid-supported solid–liquid extraction of bioactive alkaloids. II. Kinetics, modeling and mechanism of glaucine extraction from Glaucium flavum Cr. (Papaveraceae). Separation and Purification Technology 103, 279–288. ˇ Boˇzin B, Kladar N, Grujic´ N, Anaˇckov G, Samojlik I, Gavaric´ N, Coni c´ BS, 2013. Impact of origin and biological source on chemical composition, anticholinesterase and antioxidant properties of some St. John’s Wort species (Hypericum spp., Hypericaceae) from the central Balkans. Molecules 18, 11733–11750. Bruni R, Sacchetti G, 2009. Factors affecting polyphenol biosynthesis in wild and field grown St. John’s Wort (Hypericum perforatum L. Hypericaceae/Guttiferae). Molecules 14, 682–725. Cacace JE, Mazza G, 2002. Extraction of anthocyanins and other phenolics from black currants with sulfured water. Journal of Agricultural and Food Chemistry 50, 5939–5946. Cacace JE, Mazza G, 2003. Optimization of extraction of anthocyanins from black currants with aqueous ethanol. Journal of Food Science 68, 240–248. Caccia S, 2006. Main active components of St. John’s Wort (Hypericum perforatum) extracts: current analytical procedures for pharmacokinetics and concentration–response studies. Current Pharmaceutical Analysis 2, 59–68. Danila AO, Gatea F, Radu GL, 2011. Polyphenol composition and antioxidant activity of selected medicinal herbs. Chemistry of Natural Compounds 47, 22–26. Elson T, 2007. Concepts of fluid flow. In: Simons SJR (Ed.), Concepts of Chemical Engineering 4 Chemists. RSC Publishing, Cambridge.

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