food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754
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Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Extraction of phenolic compounds from Vitex agnus-castus L. Mohamed Latoui a,b , Bahar Aliakbarian b , Alessandro A. Casazza b , Mongi Seffen a , Attilio Converti b , Patrizia Perego b,∗ a b
Laboratory of Chemistry, Higher Institute of Agronomy, Chott Meriam 4042, Sousse, Tunisia Department of Chemical and Process Engineering “G.B. Bonino”, Genoa University, via Opera Pia 15, I-16145 Genoa, Italy
a b s t r a c t The primary objective of this study was to valorized Vitex agnus-castus residues in terms of phenolic compounds. The effects of extraction time (30–360 min), solid to liquid ratio (0.1–0.3 gDryBiomass /mlSolvent ), type of solvent and different tissue types (leave, roots and seeds) on total polyphenols, o-diphenols, total flavonoids and anthocyanins were evaluated. The highest total polyphenol (31.5 mgCaffeicAcidEquivalent /gDryBiomass ) and odiphenol (12.4 mgCaffeicAcidEquivalent /gDryBiomass ) contents were obtained from methanolic extract of leaves after 180 min using a solid/liquid ratio of 0.1 gDryBiomass /mlSolvent , while total flavonoids, reached a maximum value of 19.4 mgCatechinEquivalent /gDryBiomass after 360 min under the same conditions. Roots of V. agnus-castus were found to be a good source of anthocyanins with the highest yield of 0.62 mgMalvidinEquivalent /gDryBiomass using ethanol as a solvent (180 min and 0.2 gDryBiomass /mlSolvent ). The maximum antiradical power (178.5 lextract /gDPPH ) was exhibited by the methanolic leave extract obtained after 360 min at solid/liquid ratio of 0.3 gDryBiomass /mlSolvent . © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Vitex agnus-castus; Antioxidant recovery; Solid–liquid extraction; DPPH
1.
Introduction
Vitex agnus-castus (VAC), also note as chaste tree, is an important medicinal plant belonging to the Verbenaceae family, that grows naturally in Mediterranean area and is widespread in South, West and North Turkey (S¸arer and Gökbulut, 2008). In Turkish folk medicine, VAC is used as diuretic, digestive, antifungal, anti-anxiety, early birth and stomachache (Honda et al., 1996). Ripe fruit of VAC is used in folk medicine for the treatment of various obstetric and gynecological disorders, and in ancient Greek times it was also for the treatment of menstrual problems, pain, swelling, inflammation, headaches, rheumatism, and sexual dysfunction (Webster et al., 2006). Every plant is a complex of wide range of chemical moieties, among which alkaloids, polyphenols, terpenes, steroids, essential oils and glycosides. These secondary metabolites are synthesized by various metabolic pathways, which can be used by plants for protection against yeast and moulds as well as resistance to insect attack, and in some of them even for protection against ultraviolet and drought conditions (Dweck,
∗
2009). These metabolites were detected for multiple uses in herbal medicine, pharmacology, cosmetics, food, etc. Their composition may vary according to the area of growth, soil and weather conditions, harvest period, cultivation process and, of course, to the plant part (leaves, seeds, etc.) (Gairola et al., 2010; Salick et al., 2009). The storage conditions, extraction time and type of solvent are considered to have a significant impact on the final chemical composition. For instance, the content of natural preservatives produced by plants to protect the fruit and the leaves will fall dramatically once they have been separated from the main plant (Dweck, 2009). The productivity of secondary metabolites may be also enhanced by optimizing cultivation conditions, cell selection and genetic transformation (Kovalenko et al., 2004). In the last few years, the search for non-aggressive, alternative hormonal therapies with little or no side effects has become of clinical importance, and the influence that VAC exerts on the female hormonal system has received considerable attention (Lucks et al., 2002). Most of the clinical studies have been performed using alcoholic extracts of the mature
Corresponding author at: Via Opera Pia 15, 16154 Genoa, Italy. Tel.: +39 010 3532916; fax: +39 010 3532586. E-mail address:
[email protected] (P. Perego). Received 16 August 2011; Received in revised form 16 January 2012; Accepted 19 January 2012 0960-3085/$ – see front matter © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2012.01.003
food and bioproducts processing 9 0 ( 2 0 1 2 ) 748–754
fruits (Batchelder and Scalzo, 1995); therefore, a considerable amount of researches has been carried out to verify the pharmacological activity and to investigate the modality of VAC extract action. Extract with non-particular side effects became one of the most popular alternative therapies for women who do not respond to or tolerate the hormonal or psychotropic drugs (Schellenberg, 2001; Webster et al., 2006). In the last decades, several successful clinical trials supported the use of VAC extracts for the treatment of premenstrual syndrome (PMS) (Schellenberg, 2001), as the likely result of its opiate activity (Webster et al., 2006). Hirobe et al. (1994) have also reported that the extract of VAC fruits, ripened in Israel, exerts an antitumor effect on the Chinese hamster lung carcinoma cells line V-79. Moreover, VAC methanol extracts were shown to contain eight flavonoids, seven of which having cytotoxic activity against mouse lymphocytic leukemia P388. Since VAC is a medicinal plant, it is essential to quantify and qualify its active compounds that contribute to therapeutic and medicinal activities. Several studies have reported that VAC contains a lot of different secondary metabolites, among which iridoids (Hänsel and Winde, 1959), flavonoids (Hirobe et al., 1997), diterpenoids (Li et al., 2002), essential oils ˇ et al., (Kuruüzüm-Uz et al., 2003), ketosteroids (Saden-Krehula 1990). Since there is no report, to the best of our knowledge, on phenolic compounds extraction from VAC grown naturally in Tunisia and their characterization, an accurate study to qualify and quantify alcoholic extracts from such a medicinal plant would be of great industrial concern. Based on these considerations and with the aim of exploiting the potential of Tunisian VAC residues from the pharmaceutical industry, the present study was addressed to conventional phenolic extraction from different parts (leaves, roots and seeds) of this plant, selecting the extraction time (30–360 min), solid to liquid ratio (0.1–0.3 gDB /mlS ) and type of solvent (ethanol or methanol) as the independent variables, and the levels of total polyphenols, o-diphenols, flavonoids and anthocyanins as the responses. The antioxidant activity of leave extracts was also investigated in terms of their ability to reduce the radical 2,2-diphenyl-1picrylhydrazyl (DPPH).
2.
Materials and methods
2.1.
Reagents
Analytic grade methanol and ethanol, Folin–Ciocalteau reagent and pure standards of caffeic acid, catechin, and malvidin were employed. All these reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Standard stock solutions were prepared with methanol or ethanol, wrapped in aluminium foil and stored at −20 ◦ C prior to analyses.
2.2.
Raw material
In this work, we used VAC grown naturally in the Tunisian regions of Oued Barkoukech (east of Tabarka) and Ichkeul Lake (near city of Bizerte). Different parts of VAC (leaves, roots and seeds) were collected from fresh plants. Samples were dried in room temperature (20 ± 1 ◦ C), then ground to a fine powder (particle size of 0.8 mm) using a laboratory mixer, and stored at 4 ◦ C. All samples were analyzed within 3 months after collection.
2.3.
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Extraction method
Dried and milled samples of seeds, leaves or roots were extracted in glass test tubes with screw caps, and the suspension was continuously mixed by a magnetic stirrer, model Mr 2002 (Heidolph, Kelheim, Germany). The extractions were performed in the dark and at room temperature using a solid–liquid ratio of 0.10, 0.20 and 0.30 grams of dried biomass per milliliter of solvent (gDB /mlS ), and the extraction time was 30, 60, 90, 180, 360 and 420 min. Tests were performed in duplicate varying the amount of dry samples, while the solvent volume was kept constant to maintain the same head space in the test-tubes in order to prevent oxidation reactions. The extract was separated from solid fraction by centrifugation at 6000 × g for 10 min (ALC PK131 Centrifuges, Alberta, Canada) and then subjected to quantitative analyses. The solid/liquid ratio (S/L) is usually considered a factor having a large impact on the extraction of phytochemicals from various plant matrices, and the range selected in the present study was based on values frequently reported in the published literature (Casazza et al., 2010). Methanol and ethanol were selected as extraction solvents as they have been frequently used to extract phenolic compounds from different plants (Bukhari et al., 2008; Webster et al., 2006). In order to compare the yield of phenolic compounds extracted from seeds, leaves or roots at room temperature with the extraction at higher temperatures, additional extractions were performed using Soxhlet apparatus (Sarikurkcu et al., 2009). The dried samples (2 g) were extracted by using a Soxhlet extractor for 5 h with 100 ml of deionized water. The water extracts where then filtered through 0.20 m membranes (Sartorius Stedim Biotech GmbH, Goettingen, Germany) and kept at 4 ◦ C for further analysis. Extractions were performed in duplicate.
2.4.
Total polyphenols
Total polyphenol (TP) concentration was measured by the colorimetric Folin–Ciocalteau assay (Swain and Hillis, 1959) using a UV–Vis spectrophotometer, model Lambda 25 (Perkin Elmer, Wellesley, MA, USA) at a wavelength of 725 nm. TP concentration was calibrated (R2 = 0.999) using standard methanolic or ethanolic solutions of caffeic acid (10–1000 g/ml), and expressed as milligrams of caffeic acid equivalent per gram of dry biomass (mgCAE /gDB ) by means of the linear relationship: ABS725 = 0.002TP + 0.004
(1)
In fact, caffeic acid has been widely used as a reference when working with TP extracts (Aliakbarian et al., 2008, 2009; Ranalli et al., 2001). Analysis were performed in triplicate.
2.5.
o-Diphenols
The concentration of o-diphenols (OD) in the alcoholic extract, also expressed as mgCAE /gDB , was determined by the molybdate method (Gutfinger, 1981). Analysis were performed in triplicate. The calibration straight line (R2 = 0.999) was made with standard alcoholic solutions of caffeic acid in the range 10–250 g/ml: ABS350 = 0.004OD + 0.001
(2)
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Total flavonoids
The total flavonoids (TF) content of the alcoholic extracts was determined using the colorimetric method described by Jemai et al. (2009) with some modifications. After mixing 0.25 ml of each dilute extract with 1.25 ml of deionized water, we added 0.075 ml of 5% sodium nitrite solution, after 5 min of reaction 0.15 ml of 10% aluminium chloride, after additional 6 min 0.5 ml of 1.0 M NaOH, and finally deionized water until a final volume of 3 ml. The absorbance of the mixture was determined at 510 nm using the same spectrophotometer as above. Analysis were performed in triplicate. The calibration straight line (R2 = 0.991) was made using standard alcoholic solutions of catechin in the range 0.01–0.50 g/ml: ABS510 = 0.002TF + 0.012
2.7.
(3)
Anthocyanins
In order to determine the anthocyanins (AN) content of the alcoholic extracts, the colorimetric method described by Di Stefano et al. (1989) was used. According to this method, 1 ml aliquot of extract was diluted with a 70:30 (v/v) ethanol/distilled water solution, and 1 ml of 1.0 M HCl was added. The solution was mixed thoroughly, and the absorbance of the mixture determined at 540 nm. The AN content was calculated by the following equation: AN = 16.17ABS540 D
(4)
where D is the dilution factor, and expressed as milligrams of malvidin equivalent per gram of dry biomass (mgME /gDB ). Analysis were performed in triplicate.
promoted by oxygen present in the empty space of test tubes (Liyana-Pathirana and Shahidi, 2005; Yap et al., 2009).Also, the yield of phenolic compounds decreased with increasing S/L (0.10, 0.20 and 0.30 gDB /mlS ) in all fractions, because the larger the volume of solvent, the higher the phenolic compounds fraction solubilized (Prasad et al., 2009).
3.1.
Total polyphenols and o-diphenols
Figs. 1 and 2a–c show the results of the quantitative determinations of the various fractions of TP and OD. Making reference to the optimal extraction conditions, we can see in Fig. 1a, that TP extraction from leaves was more effective with methanol (31.5 mgCAE /gDB ) than with ethanol (20.5 mgCAE /gDB ), although both occurred after the same extraction time (180 min) and S/L (0.10 gDB /mlS ). These values are higher than the TP contents of acetone extracts from star fruit (Averrhoa carambola L.) residues (9.6 mg/gDB expressed as gallic acid-GA) (Yap et al., 2009) and methanolic extract from olive oil (0.6 mgCAE /gDB ) (Aliakbarian et al.,
a Total Polyphenols (mgCAE/gDB)
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3.
Results and discussion
The results of the quantitative analyses of TP, OD, TF and AN in the leave, seed and root extracts from VAC, which are illustrated in Figs. 1–4, show that the yields of these chemical compounds were greatly influenced either by the extraction time or the solid/liquid ratio (S/L). In general, the yield of any class of compounds increased with the extraction time up to a maximum value and then decreased with further prolonging the extraction time. This was the likely result of well-known oxidative phenomena
b Total Polyphenols (mgCAE/gDB)
The antioxidant activity of the extracts was measured in terms of hydrogen-donating or radical-scavenging ability by means of the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH• ) method (Brand-Williams et al., 1995), which is widely used to describe the antiradical power of different matrices (Aliakbarian et al., 2009). For each sample, seven different dilutions ranging from 1:2 to 1:128 in methanol or ethanol were prepared. For each extract, 0.10 ml of diluted sample was mixed with 3.90 ml of DPPH• methanolic solution (9.15 × 10−5 mol/l). The reaction mixtures were shaken and incubated for 1 h in the dark at room temperature, and then the absorbance was read at 515 nm using the same spectrophotometer as above. The antiradical power (ARP gDPPH /lextract ) was expressed as 1/EC50, where EC50 is the amount of extract necessary to decrease the initial DPPH• concentration by 50%. Analysis were performed in triplicate.
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Time (min) Fig. 2 – Results of o-diphenols extraction as function of time (min) using ethanol and methanol as solvents: (a) leaves; (b) seeds; (c) roots.
Fig. 3 – Results of total flavonoids extraction as function of time (min) using ethanol and methanol as solvents: (a) leaves; (b) seeds; (c) roots.
2008), but lower than that in ethanolic extracts of grape seeds (73.7 mgGAE /gDB ) (Casazza et al., 2010) and methanolic extracts of olive pomace (45.2 mgCAE /gDB ) (Aliakbarian et al., 2011). The above behavior was qualitatively similar to that of OD present as the only fraction of simple phenols (Fig. 2, panel a). These compounds, which were found to have special antioxidant activity and were reported to play an important role in extract stability (Aliakbarian et al., 2008), reached, under the same conditions, maximum values of 7.4 mgCAE /gDB in ethanol and 12.4 mgCAE /gDB in methanol, respectively, which constituted 29.4–46.9% of TP, with only a few differences depending on the solvent used. Surprisingly, ethanol behaved as a better solvent than methanol to extract from seeds the same classes of compounds (Figs. 1 and 2, panels b), whose concentrations in the extract reached after only 30 min maximum values (19.2 mgCAE /gDB TP and 5.4 mgCAE /gDB OD) about 3–5-fold those obtained with methanol. Because of the very low TP and OD contents of roots, no significant variation in solvent selectivity was detected, the highest values for both classes of compounds
(2.8–2.9 mgCAE /gDB TP and 1.3–1.9 mgCAE /gDB OD) having been obtained again at S/L = 0.10 gDB /mlS (Figs. 1 and 2, panels c). In general, the highest yields from all three plant parts were obtained at the lowest S/L (0.10 gDB /mlS ), which means that the solvent amounts employed at higher S/L were insufficient to complete the extraction of these two classes of compounds. Although the TP and OD yields followed the above-mentioned general increase with time up to maximum values, and subsequent decrease due to degradation, the time of maximum yields seemed to strongly depend on the plant part. It did in fact increase from 30 min with seeds to 180 min with leaves, as the likely result of the different ability of solvent to reach the inside of cells consequent to the different susceptibility of these plant organs to the milling operation.
3.2.
Flavonoids and anthocyanins
As is well known, the flavonoids are a class of plant secondary metabolites or yellow pigments having structures similar to those of flavones. Total flavonoids in both ethanolic and methanolic extracts represented the major fraction of TP,
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varying in the range 55.2–69.1% in leaves and 30.2–63.2% in roots, thus confirming their abundance in V. agnus-castus reported in the literature (Hoberg et al., 2000). Consistently with the more hydrophobic structure of their molecules, TF extraction from leaves required in methanol longer extraction time compared to TP, reaching a maximum value of their concentration in the extract (19.4 mgCE /gDB ) after no less than 360 min, while ethanol ensured less efficient extraction (12.8 mgCE /gDB after 180 min) (Fig. 3). From seeds, likewise TP and OD, the maximum TF yield (14.6 mgCE /gDB ) with ethanol was about 3.5-fold that with methanol and was detected only after 30 min, confirming the less recalcitrant
nature of this plant organ with respect to leaves. Finally, although the very low TF concentrations in root extracts (close to the detection limit of the analytical procedure) do not allow highlighting any appreciable influence of the other two operating variables, it is clear that methanol behaved as a much better solvent than ethanol, providing a maximum level in the extract of 1.69 mgCE /gDB after180 min. Anthocyanins are the most important pigments of the vascular plants with potential antioxidant activity. Several reports focused on the biological activity (Kong et al., 2003) and the effect of this important class of flavonoids in the prevention of cardiovascular diseases and diabetes and cancer treatment (Nichenametla et al., 2006). The results of extraction tests made on all three plant parts (Fig. 4a–c) show that, contrary to the other classes of compounds, their concentration was the highest in the root extract (0.56, 0.62 and 0.48 mgME /gDB with 0.3, 0.2 and 0.1 gDB /mlS , respectively, with ethanol and 0.36 mgME /gDB with methanol) and almost negligible in the seed one (<0.12 mgME /gDB with both solvents). This behavior might be due to the fact that anthocyanins are highly instable and very susceptible to degradation induced by light, oxygen and temperature. In this aspect, Laleh et al. (2006) found that exposure to the light accelerated the destruction of anthocyanins from four different Berberies species up to 26% when compared with dark conditions. They also confirmed that temperatures up to 35 ◦ C resulted to anthocyanins’ degradation. One interesting finding that can be gleaned from these results is that this family of flavonoids constituted only a very little fraction of TF both in leaves (1.17–4.02%) and seeds (0.77–6.99%) extracts, almost irrespectively of the extraction solvent, whereas it accounted for even 38.3 and 31.5% of TF extracted from roots with ethanol and methanol, respectively.
3.3.
Soxhlet extraction
In order to ensure complete extraction of phenolic compounds, the aqueous Soxhlet extraction was employed using different parts of biomass. The results reported in Table 1 demonstrated the same trends for TP, OD and TF when using leaves, roots and seeds. While in the case of AN, higher content was achieved when using seeds as biomass followed by roots and leaves. The comparative study showed that the highest TP and OD contents (31.50 and 12.36 mgCAE /gDB ) which were obtained using leaves, methanol and 180 min were lower than the Soxhlet extract results (46.85 and 43.44 mgCAE /gDB for TP and OD, respectively). Using alcoholic extraction, in the same operative conditions and longer extraction time (360 min) TF resulted to be 19.41 mgCE /gDB , this value was approximately the half that obtained by Soxhlet apparatus (42.29 mgCE /gDB ). In the case of roots, alcoholic extraction resulted to be more efficient for AN recovery in comparison with aqueous Soxhlet extraction.
Table 1 – Results of total polyphenols (TP), o-diphenols (OD), total flavonoids (TF) and anthocyanins (AN) obtained by Soxhlet extraction of leaves, seeds and roots of Vitex agnus-castus using water. TP (mgCAE /gDB ) Leaves Seeds Roots
46.85 ± 1.42 25.87 ± 0.91 8.81 ± 0.47
OD (mgCAE /gDB ) 43.44 ± 2.10 18.42 ± 0.27 6.45 ± 0.14
TF (mgCE /gDB )
AN (mgME /gDB )
42.29 ± 1.86 16.00 ± 0.81 2.92 ± 0.28
0.38 ± 0.01 0.60 ± 0.01 0.52 ± 0.01
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The authors are grateful to the Erasmus Mundus Program IMAGEEN for the PhD fellowship of Mohamed Latoui.
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Time (min) Fig. 5 – Antioxidant activity expressed as antiradical power (ARP) of (a) ethanolic and (b) methanolic extracts of VAC leaves. These differences might be due to the fact that higher temperatures (100 ◦ C) in the case of using Soxhlet enhanced solvent solubility and consequently higher phenolic extraction yields.
Antioxidant activity
An evaluation of the antioxidant activity of V. agnus-castus extracts was done using exclusively the methanolic extracts of leaves, since they exhibited by far the highest total polyphenols yield (31.5 mgCAE /gDM ). It is well known in fact that this activity is directly related to the antiradical power of these compounds (see Section 2).The results illustrated in Fig. 5 show that the extraction time considerably increased the ARP up to a maximum value, beyond which it decreased owing to oxidation. However, as far as the effect of S/L is concerned, this operating variable had, as expected, an opposite effect compared to the previous responses (TP, TF, etc.), because the most concentrated the extracts (less efficient extraction), the highest their ARP. In particular, the maximum ARP (178.5 lextract /gDPPH ) was exhibited by the methanolic leave extract obtained after 360 min at S/L = 0.3 gDB /mlS . Such an ARP yield was 76.6% higher than the one detected in the best ethanolic extract. This value is much higher than those (0.27 lextract /gDPPH ) obtained by Hajdú et al. (2007) for VAC fruit extracts, either using ethylacetate (14.7 l/g) or methanol/water (0.3 l/g) as solvent.
4.
Acknowledgment
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Conclusions
In this work we characterized for the first time different parts (leaves, seeds and roots) of Tunisian V. agnus-castus in terms of phenolic compounds (total polyphenols, o-diphenols, total flavonoids and anthocyanins). The highest total polyphenol, o-diphenol and flavonoid contents were obtained from methanolic extract of leaves. Roots of Vitex were found to be a good source of anthocyanins with the highest yield using ethanol as solvent. Methanolic leave extracts exhibited an interesting antioxidant activity. The preliminary results achieved in this part of the study suggest that further complete optimization of the proposed treatment, taking into account the effects of extraction temperature, drying time and qualification of the single phenolic compounds in the extracts, will be necessary. Naturally grown VAC can be successfully used as an inexpensive source of phenolic compounds, and the food and pharmaceutical industries may be benefited.
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