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Piperine-rich extracts obtained by high pressure methods
Accepted Manuscript Title: PIPERINE-RICH EXTRACTS OBTAINED BY HIGH PRESSURE METHODS Authors: K´atia S. Andrade, Guilherme Trivellin, Sandra R.S. Ferreira PII: DOI: Reference:
S0896-8446(17)30181-X http://dx.doi.org/doi:10.1016/j.supflu.2017.05.001 SUPFLU 3912
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
13-3-2017 26-4-2017 1-5-2017
Please cite this article as: K´atia S.Andrade, Guilherme Trivellin, Sandra R.S.Ferreira, PIPERINE-RICH EXTRACTS OBTAINED BY HIGH PRESSURE METHODS, The Journal of Supercritical Fluidshttp://dx.doi.org/10.1016/j.supflu.2017.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PIPERINE-RICH EXTRACTS OBTAINED BY HIGH PRESSURE METHODS
Kátia S. Andrade1, Guilherme Trivellin1, Sandra R. S. Ferreira1*. 1
Chemical and Food Engineering Department – Federal University of Santa Catarina – EQA/UFSC – C.P. 476, CEP 88040-900, Florianópolis, SC, Brazil.
*
Corresponding author: [email protected] (S.R.S. Ferreira).
GRAPHICAL ABSTRACT
HIGHLIGHTS
Black pepper extraction with ethanol provides a product with high antioxidant potential;
SFE is suitable technology to provide extracts with high quality in terms of chemical composition and solvent free;
PLE, SFE and LPE extracts of black pepper have piperine and caryophyllene as major compounds.
PLE technique is a promising method to obtain bioactive extracts from black pepper.
ABSTRACT
Chemical composition and antioxidant activity of black pepper extracts were studied. The high pressure methods Supercritical Fluid Extraction (SFE) (at 40, 50 and 60 °C and from 150 to 300 bar) and pressurized liquid extraction (PLE) with ethanol were compared to Soxhlet (SOX) and ultrasound-assisted extraction (UAE). The comparison considered process yield, extract composition and antioxidant activity, evaluated by DPPH (1,1diphenyl-2-picrylhydrazil) and ABTS [2,2-azino-bis (3-ethylbenzotiazoline-6-sulphonic acid)] methods, and total phenolic content by Folin-Ciocalteau procedure. Highest global yield (X0 = 10.3 ± 0.6 %) and antioxidant potential was obtained by SOX extract with ethanol. The chemical profile was evaluated by CG-MS analysis. The main compound identified was piperine for the supercritical and ethanolic extracts. Despite the several researches concerning SFE of black pepper, the importance of this raw material justifies a broader understanding of this material, added to the novelty of PLE and ABTS procedures applied for black pepper material.
Keywords: Supercritical technology; piper nigrum; piperine, antioxidant activity.
1. Introduction The black pepper plant is the most commonly used spice in the world due to its commercial, economical, nutritional, and medicinal value. The seeds are commonly used to enhance the taste and flavor of food, while the essential oil plays a significant role in food processing, health, and nutrition by preventing or delaying spoilage of foods due to its antioxidant and antimicrobial properties [1].
Brazil is among the three largest producers of peppers in the world and has about 90 % of its production exported [2]. The components of the pepper’s extract that contribute to its value as a food additive are the volatile oil for its aroma and the alkaloid compounds for the pungency. Black pepper is used in skin care, muscle and joint pains, and in improving blood circulation and respiratory systems [3]. Piperine, the major component present in black pepper, is an alkaloid responsible for pungency and irritation and it has demonstrated antioxidant and antimicrobial properties. This alkaloid is an alternative and complementary treatment to various disorders [4], has pharmacological impact on nervous and neuromuscular systems, and exercises sedative effect and helps in digestion [3]. Ecofriendly techniques of extraction and recovery of bioactive compounds are very attractive in the last years. Among these green technologies, the extraction assisted by ultrasound, by pressurized liquid and extraction using supercritical fluids must be highlighted [5–7]. Ultrasound-assisted extraction (UAE) is based on the use of the energy of sound waves that provoke expansion and compression cycles in a medium (mechanical vibration). This phenomena accelerates the extraction of compounds in reduced time due to the influence on the mass transport, solvent/sample ratio and temperature [8]. Pressurized liquid extraction (PLE) consists of the continuous solvent flow through a fixed solid bed of particles placed inside the extraction column. The main advantages of PLE are higher extraction efficiencies requiring lower amounts of solvents, reduced extraction time and the use of solvents classified as Generally Recognized as Safe (GRAS) [9–11]. Supercritical fluid extraction (SFE) is a green process and gained attention over traditional methods because it protects against thermal degradation and solvent contamination of the extracts [12].
Therefore, this study proposes to obtain natural extracts from black pepper (piper nigrum) in order to evaluate the application of green technologies in the recovery of compounds of high added value, by analyzing the composition profile and the antioxidant activity of the extracts. Black pepper, as the most important spice commodity, presents particular characteristics that always raise academic interest, either concerning the crop or the processing differences. Also, to the bests of our knowledge and to broad the use of green methods to obtain black pepper extracts, no PLE research was detected. Therefore, although there are several researches concerning SFE of black pepper, the importance of this raw material justifies a broader understanding of this material, together with the innovation regarding the PLE and ABTS procedures applied for black pepper material.
2. Materials and methods
2.1 Raw material and sample preparation The unripe fruits of Piper nigrum L. cultivar Bragantina was gently supplied by a Farm located at São Mateus City (Espírito Santo, Brazil) (18° 43’ 37” S; 40° 05’ 51” W; 26 m of altitude). The plant was identified and a voucher specimen (number 15422; 11/18/2014) was deposited at Municipal Herbarium collection (São Paulo, Brazil). The raw material provided presented 15 ± 1 % (w/w) of moisture and volatile content, determined according to the 950.46B method of AOAC [13]. The black pepper samples were stored at room temperature (± 25 °C) and used as supplied, avoiding a previous drying step that could cause degradation in the raw material. The pepper grains were ground in a knife mill and characterized by size classification in a vertical vibratory sieve shaker. The mean particle diameter, calculated by mean size distribution as described by Gomide [14], indicated a mean value of 570 ± 1 µm. The grounded material was stored
at – 18 °C until the extractions were performed. All solvents used in the experiments were of analytical grade (PA) purchased from Lafan Química Fina LTDA. Carbon dioxide (99.9 % purity) was purchased from White Martins S/A (São Paulo, Brazil) and delivered at pressure up to 60 bar.
2.2 Extraction methods
2.2.1 Supercritical fluid extraction (SFE) A high-pressure unit, previously described by Zetzel et al. [15], was used for the SFE with CO2 by following the extraction procedure from Michielin et al. [16] to obtain the black pepper extracts. A co-solvent pump was connected to the extraction line in order to supply the modifier (organic solvent at high-pressure). Briefly, the extraction consisted of placing a fixed mass of 20 g of the dried raw material (grinded black pepper) inside the extractor cell to form the fixed bed of particles, followed by the control of the process variables (temperature and pressure). The extraction was then performed and the solute collected in amber flasks and weighed on an analytical balance (OHAUS, Model AS200S, NJ, USA). The SFE with CO2 was conducted at temperatures of 40, 50 and 60 °C and pressures of 150, 200 and 300 bar, at constant solvent flow rate of 8 ± 2 g min-1. The extraction time was set at 4 h according to the kinetic extraction curve, performed at 200 bar, 50 °C (intermediary conditions of extraction) and 8 ± 2 g CO2 min-1. At the end of this period, most of the extractable solute mass had been recovered. The solvent density values were obtained according to Angus et al. [17]. The co-solvent (CS) assays were performed at 150 bar and 50 °C at constant solvent flow rate of 8 ± 2 g min-1. Ethanol (EtOH) was used as modifier in concentrations of 2.5, 5.0 and 7.5 % (w/w) related to the CO2 mass. The co-solvent was separated from the extract according to the procedure
described for the low pressure methods (Section 2.2.2). The results were expressed in terms of extraction global yield (X0).
2.2.2 Low pressure extractions (LPE) The low pressure extraction methods, Soxhlet (SOX) and ultrasound-assisted (UAE), were applied using three different solvents: hexane (Hx), ethyl acetate (EtOAc) and ethanol (EtOH), with polarity index of 0, 4.4 and 5.2, respectively [18]. The SOX extraction was performed according to 920.39C method of AOAC [13]. Briefly, 150 mL of solvent recycling over 5 g of dried sample, in a Soxhlet apparatus for 6 h extraction conducted at the solvent boiling temperature. The ultrasound-assisted extractions consisted of placing 7 g of raw material and 210 mL of solvent inside a covered glass balloon. The extraction was performed at room temperature during 45 min. The equipment used was an ultrasonic cleaner bath (USC700/55 kHz, Unique Ltda., Indaiatuba/SP, Brazil), which operates in a frequency of 55 kHz and potency of 100 W [19]. The residual solvent from all extracts (SOX and UE with three solvents) was eliminated in a rotatory evaporator (Fisatom, 802, Brazil), supplied with cooling and vacuum control. The evaporation temperature was adjusted to a level below the solvent boiling point in order to avoid thermal degradation of the extracts. The results, expressed in extraction global yield (X0), represent the mean values ± standard deviation from duplicate experiments, for each LPE assay.
2.3 Pressurized liquid extraction (PLE) The dynamic extraction method was used in PLE with ethanol as solvent and it was carried out according to Viganó et al. [20]. This method consists of the continuous solvent
flow through a fixed solid bed of particles placed inside the extraction column. Approximately 20 g of ground black pepper was loaded in the extraction vessel, forming a fixed bed inside a stainless steel column. Then, ethanol was pressurized using a HPLC pump (Waters, EUA) until 100 bar and passed through a coil in a heating bath (Microquímica, Brazil) to reach the process temperature (40 °C). The ethanol flow rate was kept constant at 3 mL min-1 and the extraction time was set in 60 min. The extracts were collected in glass flasks, the solvent was removed following the procedure described in Section 2.2.2 and then, the dried extract was stored at -18 °C until the analysis were performed. The experiments were carried out in duplicate and global yield was expressed by mean ± standard deviation. These assays were performed preliminarily to evaluate the PLE feasibility to obtain extracts with antioxidant potential, although, complementary studies (ongoing research) may provide insights related to the influence of the PLE operational conditions and solvent types in extract quality.
2.4 Determination of total phenolic content (TPC) The TPC was determined according to the Folin-Ciocalteu spectrophotometric method [21]. Briefly, the reaction mixture was composed by 0.1 mL of extract (concentration of 1667 mg/L), 7.9 mL of distilled water, 0.5 mL of Folin–Ciocalteu reagent (a mixture of phosphomolybdate and phosphotungstate) and 1.5 mL of 20 % sodium carbonate, placed in opaque flasks. The flasks were agitated and allowed to stand for 2 h, and the absorbance was measured at 765 nm in a spectrophotometer (Femto, 800XI, Brazil). The TPC was calculated according to a standard curve (y= 0.0011x + 0.0352; R²=0.9951), previously prepared with gallic acid as standard (galic acid equivalent: GAE). The analysis was performed in triplicate and the results expressed as mg GAE g-1 extract as mean ± standard deviation.
2.5 Antioxidant potential The antioxidant potential of the black pepper extracts, obtained by different extraction methods (SFE, SFE-CS, SOX and UAE), was evaluated by two scavenging activity procedures, DPPH· and ABTS methods. The results were compared with the antioxidant activity from the synthetic compound BHT (butylated hydroxytoluene). The antioxidant potential of the PLE extract was only evaluated by DPPH· method.
2.5.1 Free radical scavenging activity (DPPH·) The free radical scavenging of black pepper extracts was evaluated using 1,1diphenyl-2-picrylhydrazil (DPPH·) as described by Mensor et al. [22]. Briefly, each extract was mixed with a 0.3 mM DPPH· ethanolic solution to give final concentrations of 5, 10, 25, 50, 125, 250 and 500 µg extract mL-1 DPPH· solutions. After 30 min at room temperature, the absorbance values were measured at 517 nm and converted into percentage of antioxidant activity (% AA). This activity was also presented as the effective concentration at 50 % (EC50), i.e., the concentration of the test solution required to give 50 % decrease in the absorbance of the test compared to that of a blank solution, and expressed in µg of extract/mL DPPH·. The EC50 values were calculated from the linear regression of the % AA curves obtained for all extract concentrations. The % AA and EC50 for all extracts were obtained considering the mean value of triplicate assays. This method was also used to describe the antioxidant potential of extracts obtained by PLE method.
2.5.2 ABTS radical scavenging assay
The ABTS [2,2-azino-bis (3-ethylbenzotiazoline-6-sulphonic acid)] assay was carried out according to Re et al. [23] and the ABTS activity was calculated considering the standard concentration curve. The synthetic vitamin E, Trolox (6-hidroxy-2,5,7,8tetramethylchroman- 2-carboxylic acid) was used as antioxidant reference, which was prepared in ethanol and stored as standard solution. Briefly, the ABTS was dissolved in water up to a concentration of 7.0 mM, and subjected to reaction with potassium persulphate (2.45 mM) for the radical formation. The ABTS solution was diluted with ethanol to absorbance of 0.70 ± 0.05 at 754 nm. Solutions were prepared with 20 μL of the extract at concentrations of 100, 250, 500 and 1000 mg mL-1, in triplicate. Than 980 μL of ABTS solution was added to the test tubes and after 6 minutes the absorbance was read at spectrophotometer (Femto, 800XI, Brazil). Results were expressed as MTEAC/g (Trolox Equivalent Antioxidant Capacity per g extract), such as mean ± standard deviation.
2.6 Chemical profile The identification and relative quantification of the volatiles compounds present in the black pepper extracts were performed by a gas chromatograph equipped with a mass spectrophotometer (GC/MS, model 7890 A, mass detector 5975C, Agilent Technologies, USA), attached to a HP-5MS column (30 m x 0.25 mm internal diameter x 0.25 µm film thickness, Agilent Technologies, USA). The carrier gas was helium with flow rate of 1 mL min-1, split ratio of 1:50, injector temperature of 250 °C and Thermal Aux 2 (MSD Transfer Line) 250 °C, while column temperature programmed from 70 to 280 °C at a rate of 3 °C min-1. The main components of black pepper extracts were identified by comparing the mass spectra and the retention time with NIST 11 Mass Spectral Library available on equipment.
2.7 Statistical analysis The global yield (X0), TPC and the potential antioxidant results were evaluated statistically by software SAS for Windows version 6.0, at 5 % level of significance, in order to identify significant differences between values of global yield, as a function of temperature, pressure, solvent/extraction type, and percentage of antioxidant activity. All analyses were carried out in triplicate and the results were expressed as means ± standard deviation (SD).
3 Results and discussion
3.1 Global yield (X0) of LPE, SFE and PLE The global yield (X0), defined as the amount of solute extractable by the solvent at the extraction conditions, indicates, quantitatively, the process efficiency. The yield results obtained for the different extraction methods and solvents (Soxhlet, UAE, SFE with CO2 and PLE) for black pepper are presented in Table 1.
Table 1. Global yield (X0) of black pepper extracts obtained by low pressure extractions (LPE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE).
The results presented in Table 1 indicate that the best yield was obtained by the Soxhlet extraction using ethanol as solvent. It is possible to observe a yield increase trend due to the increase in solvent polarity, suggesting that compounds present in plant matrix have intermediate to high polarity. Moreover, the dielectric constant may explain the superior performance of the extraction with ethanol compared to other solvents. The
dielectric constant is the ability to attenuate electric fields and thus assist in the interaction between charges. Thus, as this property has a value significantly higher for the ethanol, the solvent molecules are strongly linked to the polar components contributing to the increased extraction yield [24]. Comparing the low pressure methods for the same solvent, it was observed that the Soxhlet extraction had higher yields compared to ultrasound-assisted leaching extraction. The operating temperature of the recycle solvent and the interactions between solvent and plant matrix, characteristic of Soxhlet extraction, may contribute to increase the solubility of compounds of different types, raising the extraction yield [25–27]. The global yield of PLE with ethanol is comparable to the ultrasound-assisted extraction using ethanol and ethyl acetate as solvents. Moreover, it is important to note that in the PLE, despite the similar extraction time between the methods, the PLE advantages are lower solvent volume and no filtration step needed. In this work, only one temperature condition was evaluated, however, the use of higher temperatures may improve the solubility of the compounds of interest and their diffusivity, a subject under investigation at the ongoing research about PLE used to obtain black pepper extract. The extract to solvent ratio (E/S) represents the effectiveness of the extraction method or conditions of pressure and temperature, for each extraction method. Therefore, higher E/S ratio represents higher extraction method efficiency because less solvent is required to obtain a given mass of extract. From the E/S values presented in Table 1 it is possible to observe the best performance of the procedure PLE with ethanol (5.88 mg mL1
), compared to ethanolic extracts from other methods, for instance, SOX provided E/S
of 3.57 mg mL-1 and UAE of 1.89 mg mL-1.
A broader comparison of the extraction efficiency among the different methods using ethanol as solvent is observed in Figure 2, which relates the extraction yield, the E/S ratio and the extraction time for the methods PLE, UAE and SOX.
Figure 2. Extraction evaluation of PLE, SOX and UAE using ethanol as solvent.
The SOX yield reached the highest value (10.3 %), compared to other methods, probably caused by the solvent recycling, which leads to the raw material exhaustion with time, reducing the extraction efficiency (E/S). On the other hand, PLE and UAE methods present process time and yield values very similar, however the PLE method provides higher process efficiency (high E/S ratio), compared to UAE and SOX. Therefore, taking into account the extraction time, the volume of solvent used and the mass of extract obtained, it is possible to suggest that the PLE method was more effective that SOX and UAE, to obtain black pepper extracts. SFE with pure CO2 at the highest condition (300 bar and 60 °C) produced yield values very close to the ones obtained by Soxhlet and ultrasound-assisted extraction with hexane, suggesting the extraction of compounds with lipophylic characteristic, more easily dissolved in nonpolar solvents. The poor performance from the non-polar solvents (CO2 and hexane) is attributed to the low fatty acids content in the black pepper ground. In the extraction with supercritical CO2, the evaluation of global yield extraction, obtained under different conditions of temperature and pressure, indicates the effect of solubility of the solvent and consequently the influence on the process yield. According to Table 1, the highest yield, in supercritical conditions, for the black pepper extracts was 3.2 ± 0.3 %, obtained for the condition of 300 bar and 60 °C. This value shows no significant difference, at level of 5 %, to the results obtained at 300 bar
and 50 °C. The results achieved by the other extraction conditions also show no significant difference among each other. The pressure increase at constant temperature enhances the extraction yield, due to the increase in the CO2 density and consequently in the solvent power [12]. Besides, the increased pressure can lead to disruptions in the plant cells, facilitating the release of compounds that were not previously available, and thus increasing the yield of the process, as reported in the literature [27,28]. The lowest yields were obtained at 150 bar, at all temperatures. The effect of temperature on extraction yield, at constant pressure, occurs by two mechanisms: the increase in process temperature increases the solubility due to increased vapor pressure of the solute and on the other hand reduces the solubility due to the decrease in density the solvent. This behavior occurs near the critical point, where small changes in temperature and/or pressure are accompanied by large variations in solvent density [12,28]. Comparing the yields obtained by supercritical CO2 with the results obtained by conventional extraction (SOX and UAE) and PLE, it is observed that the low pressure extractions and PLE using ethanol as a solvent produced higher yield values to those achieved by SFE. These results can be explained by the extraction of more polar compounds, not soluble in CO2, a nonpolar solvent. Additionally, for the Soxhlet extractions, the solvent recycling, the extended extraction time and the high amount of solvent aid the yield enhancement, compared to other methods [29,30].
3.1.1 SFE with CO2 and co-solvent (CS) In order to improve the efficiency of SFE technique by increasing the recovery of polar compounds, EtOH, a green modifier, was selected as a co-solvent. The co-solvent
and the extraction conditions were selected based on the global yield, TPC and antioxidant activity results obtained by low pressure extractions and SFE with CO2. Consequently, the extraction was performed at 150 bar and 50 °C using ethanol concentrations of 2.5, 5.0 and 7.5 % (w/w). The results obtained by the SFE with cosolvent for black pepper are presented in Figure 2.
Figure 2. Global yield (X0) of black pepper extracts obtained by SFE - CO2 with cosolvent.
The SFE with ethanol as co-solvent increased the yield values for all ethanol concentrations and conditions tested. This behavior is explained by the increase in the solubility of polar compounds in the mixture ethanol/CO2, compared to the solubility in pure CO2. Furthermore, not only the solubility of a certain component increases with the use of co-solvent, but also the number of components solubilized by the solvent, which reduces the selectivity and increases the process yield [31,32]. A larger amount of co-solvent favors solute/solvent interactions and increases the yield. Generally, the type of co-solvent has a greater impact on the extraction efficiency than its concentration, because of the type of matrix-compounds interaction and physicochemical parameters of the co-solvent. Normally, better results are achieved at higher concentrations, but in the some cases the behavior is unlike [33].
3.2 Total phenolic content (TPC) and antioxidant activity
The results for the total phenolic content and antioxidant activity are presented in Table 2, obtained for all extract samples analyzed, and compared to the results presented
by the synthetic product BHT, as standard sample. The antioxidant potential was evaluated by two scavenging methods with the radicals DPPH· and ABTS. Both radical scavenging methods used in this work are popular for determination of the antioxidant capacity of foodstuff due to the simple, rapid, sensitive, and reproducible procedures. The extracts were obtained from black pepper using different extraction methods (LPE, SFE, PLE).
Table 2. Antioxidant activity and TPC of black pepper extracts.
The values of total phenolic content obtained by SFE show no variation behavior with changes in pressure or temperature. Most of supercritical extract showed similar results and lower to that found in commercial antioxidant, BHT. The highest data for SFE was obtained at 150 bar and 50 °C using 5 % ethanol as co-solvent (46 ± 1 mgGAE/ gextract). This behavior can be explained by the increase in the solubility of polar compounds in the mixture ethanol + CO2, compared to the solubility in pure CO2 [32]. The addition of ethanol as a co-solvent in the SFE of black pepper favors the extraction of phenolic compounds, except for the 2.5 % concentration of co-solvent, when the result was statistically equal to extraction with pure CO2. Andrade et al. [26] also observed a reduction in the total phenolic content in coffee grounds extracts with increased concentration of ethanol in SFE. According Campos et al. [28], there is an optimum concentration of co-solvent to be used in the SFE to achieve better yields and better antioxidant activity. Above this concentration, which in this study is 5 %, there was a reduction in the extraction of phenolic compounds, indicative of antioxidant activity. For the LPE methods, the best results of TPC were obtained with ethyl acetate as solvent, for the Soxhlet and UAE method, reaching 55 ± 6 mgGAE/gextract and 42 ± 1
mgGAE/gextract, respectively. The SFE with ethanol (5 %) produced TPC of 46 ± 3 mgGAE/gextract, statistically equal to that obtained in the ultrasound-assisted extraction using ethyl acetate as solvent. The black pepper extracts that showed the higher values of antioxidant activity from the DPPH method were obtained by Soxhlet using ethanol (75 %) and ethyl acetate (55 %) as solvents, respectively. This result may be compared to TPC values, which indicate that its antioxidant activity may be not directly related to its phenolic content. The extract obtained by PLE showed 55 % of antioxidant activity determined by DPPH method, which suggest that PLE is a promising technique to obtain bioactive extracts. Also the increase in extraction temperature may lead to the recovery of new antioxidant compounds formed from Maillard, caramelization and thermo oxidation reactions [34] . The EC50 results, calculated from DPPH data for the supercritical extracts were above the 250 µg mL-1. Bellow this value the extract is considered a good antioxidant product [28]. The behavior of the SFE may be associated to the low amount of phenolic compounds with intermediate to high polarity, since CO2, a non-polar solvent, does not favor the solubilization of such components. Similarly to the TPC results, the addition of 5% co-solvent increased the antioxidant activity of extract compared to that obtained by CO2, due to the higher polarity of the solvent mixture (CO2 and ethanol) compared to the supercritical CO2, favoring the extraction of phenolic compounds [7]. The results from Table 2 also show that Soxhlet and UAE extraction with ethyl acetate presented the highest AA % behavior by the ABTS method, with values of 430 ± 21 µMTEAC/gextract and 306 ± 70 µMTEAC/gextract, respectively. The best antioxidant capacities for supercritical extracts were found for pressure of 150 bar and 50 °C, probably because the components responsible for antioxidant characteristics detected by
the ABTS method were present in higher concentrations. This situation suggests that the presence of ethanol affect the solubilization of compounds responsible for the antioxidant activity of the extracts, detected by this method of analysis. No earlier studies are reported in the literature regarding the ABTS assay of black pepper extract obtained by the SFECO2. Natural extracts may be more beneficial than isolated bioactive compounds, because the synergic interaction of compounds may enhance the properties of the individual components. Moreover, the use of natural extracts may be beneficial considering that maximum lawful levels for synthetic food additives are established based on various toxicological parameters which are usually not applicable to naturally occurring compounds [35,36].
3.3 Chemical profile The gas chromatography analysis was performed for the black pepper extracts in order to evaluate the volatile fraction, mainly obtained by low polar solvents, such as supercritical CO2 or hexane. The chemical profile results are presented in Table 3, with the compounds identified and the relative composition (integrated composition) for all extracts for black pepper obtained by supercritical fluid extraction (SFE), low pressure extraction (SOX and UAE) and pressurized liquid extraction (PLE). The major compounds identified in terms of area percentage relative and/or impact in different extracts were piperine and caryophyllene.
Table 3. Relative composition profile, in % peak area, of black pepper extracts.
Piperine is the alkaloid responsible for the pungency of black pepper and it has been used in many forms in traditional medicine. Several biological activities are associated with piperine, such as antioxidant, antitumor, antidepressant activity, among others [37– 39]. No trends were detected concerning the effects of pressure and temperature on composition of supercritical extracts. The identified compounds exhibit valuable biological activity, which may suggest the use of extracts in various therapeutic applications, considering, of course, the synergistic effect between the compounds. A similar composition was found by Bagheri et al. [40] and Ferreira et al. [41] for black pepper essential oil obtained by SFE at 300 and 150 bar, 40 °C, respectively. In that case, the majoritary compounds were sabinene and caryophyllene. Among the identified components, it is important to highlight the sesquiterpenes with reports of antitumor activities, such as caryophyllene [42] and humulene [43]. The extracts obtained by low polar solvents provided a wider varied of sesquiterpenes, compared to other extracts. Based on the results of extract composition and yield, two extracts obtained by SFE (200 and 300 bar, 40 °C) were submitted to the analysis of antitumor activity in the work developed by Grinevicius [44]. In vivo treatment with the black pepper SFE of isogenic Balb-c mice bearing Ehrlich ascites carcinoma showed higher inhibition and increased the survival time compared to ethanolic extract. The highest concentration of piperine and sesquiterpenes in these extracts may have caused the cytotoxicity and the inhibition of the cell proliferation through apoptosis, resulting in best antitumor performance of the supercritical extract. Although the piperine was the major compound detected in the black pepper extracts, the Piper nigrum biological activities may be a result of the synergic interaction among the different compounds present in the raw material. Thus, further
studies related to black pepper extracts may be of interest, starting from this work that target the piperine component.
4. Conclusions The use of black pepper as raw material for different extraction methods is promising due to the high quality of the substances present in this vegetable matrix. When comparing the different extraction methods, besides the determination of the process yield, it is also necessary to quantify the antioxidant potential of the product (extract) by diverse procedures and also evaluate the chemical composition of the extracts. SOX and UAE presented highest extraction yield when using ethanol as solvent. SFE extracts presented lowers yields when compared to SOX and UAE methods. The use of ethanol as co-solvent in SFE increased the yield extraction and also the antioxidant activity when 5 % of ethanol was added to the process. PLE is an emerging technology that has shown good results in relation to the extraction of bioactive compounds and need further studies in order to evaluate the influence of the different operational conditions on the extracts quality. The supercritical fluid extraction is an effective technique for the recovery of extracts rich in piperine, comparable to extraction by Soxhlet with ethanol, providing a high valueadded and solvent-free extract. Finally, it is important to point that each extraction procedure presents particular characteristics to provide products for different applications. The challenge is to explore the extraction in order to optimize the extract composition profile, related to the raw material specificities.
Acknowledgments The authors wish to acknowledge CAPES (Coordination for the Improvement of Higher Education Personnel) for the scholarship, CNPq (National Council of
Technological and Scientific Development) (Process number 473153/2012-2) for the financial support.
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pomace extracts obtained by supercritical fluids, J. Biotechnol. 164 (2013) 423– 432. doi:10.1016/j.jbiotec.2012.09.014. [31] M. Herrero, A. Cifuentes, E. Ibañez, Sub- and supercritical fluid extraction of functional ingredients from different natural sources: Plants, food-by-products, algae and microalgae - A review, Food Chem. 98 (2006) 136–148. doi:10.1016/j.foodchem.2005.05.058. [32] C.G. Pereira, M.A.A. Meireles, Supercritical fluid extraction of bioactive compounds: Fundamentals, applications and economic perspectives, Food Bioprocess Technol. 3 (2010) 340–372. doi:10.1007/s11947-009-0263-2. [33] J. Hollender, J. Shneine, W. Dott, M. Heinzel, H.W. Hagemann, G.K.E. Gotz, Extraction of polycyclic aromatic hydrocarbons from polluted soils with binary and ternary supercritical phases, J. Chromatogr. A. 776 (1997) 233–243. [34] M. Plaza, C. Turner, Trends in Analytical Chemistry Pressurized hot water extraction of bioactives, Trends Anal. Chem. 71 (2015) 39–54. doi:10.1016/j.trac.2015.02.022. [35] A.T. Borchers, C.L. Keen, M.E. Gershwini, Mushrooms, tumors, and immunity: an update, Experimental Biology and Medicine 229 (2004) 393 – 406., 229 (2004) 2004. [36] F. Bonilla, M. Mayen, J. Merida, M. Medina, Extraction of phenolic compounds from red grape marc for use as food lipid antioxidants, Food Chem. 66 (1999) 209–215. doi:10.1016/S0308-8146(99)00046-1. [37] E.S. Sunila, G. Kuttan, Immunomodulatory and antitumor activity of Piper longum Linn. and piperine, J. Ethnopharmacol. 90 (2004) 339–346. doi:10.1016/j.jep.2003.10.016. [38] J. Wattanathorn, P. Chonpathompikunlert, S. Muchimapura, A. Priprem, O.
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FIGURE CAPTIONS
Figure 1. Extraction evaluation of PLE, SOX and UAE using ethanol as solvent.
Figure 2. Global yield (X0) of black pepper extracts obtained by SFE - CO2 with cosolvent (ethanol) at 150 bar and 50 °C.
FIGURE GRAPHICS
12
10
8
6
4
2
0 PLE
UAE X0 (%)
E/S (mg/mL)
SOX Extraction time (h)
Figure 1. Extraction evaluation of PLE, SOX and UAE using ethanol as solvent.
6 5
Yield (%)
4 3 2 1 0 0
5 2.5 Co-solvent (%, w/w)
7.5
Figure 2. Global yield (X0) of black pepper extracts obtained by SFE - CO2 with cosolvent (ethanol) at 150 bar and 50 °C.
TABLES Table 1. Global yield (X0) of black pepper extracts obtained by low pressure extractions (LPE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE). Extraction Method
40 °C
SFE-CO2
50 °C
60 °C
LPE
SOX
UAE
PLE 100 bar/40 °C (1)
ρCO2(1) (g/cm³) 0.781
1.5 ± 0.4de
E/S(4) (mg/mL) 0.12
200 bar
0.840
2.1 ± 0.1cde
0.24
300 bar
0.911
2.4 ± 0.4bc
0.22
150 bar
0.701
1.28 ± 0.01e
0.10
200 bar
0.785
1.6± 0.4cde
0.14
300 bar
0.871
2.7 ± 0.1ab
0.26
150 bar
0.606
1.4 ± 0.3e
0.08
200 bar
0.724
2.2 ± 0.2bcd
0.17
300 bar
0.830
3.2 ± 0.3a
0.28
Process Parameters 150 bar
X0 (%)(2)
Solvents
SPI(3)
Hx
0
4.1 ± 0.5C
1.35
EtOAc
4.3
6.5 ± 0.3B
2.50
EtOH
5.2
10.3 ± 0.6A
3.57
Hx
0
3.1 ± 0.1E
1.04
EtOAc
4.3
5.6 ± 0.1C
1.72
EtOH
5.2
5.3 ± 0.5C
1.89
Solvents EtOH
SPI(3) 5.2
5.265 ± 0.007C
5.88
CO2 density (Angus, Armstrong & Reuck [17]). Same letters indicated no significant difference at level of 5% (p < 0.05). (3) Solvent polarity index (4) Extract to solvent ratio Hx, hexane; EtOAc, ethyl acetate; EtOH, ethanol. (2)
Table 2. Antioxidant activity and total phenolic content for black pepper extracts Extracti on Methods UAE
SOX
SFECO2
SFECO2+2.5 % EtOH SFECO2+ 5 % EtOH SFECO2+ 7.5 % EtOH PLE EtOH BHT(6) (1)
Process Paramete rs Hx
TEAC(4)(μMTEA C/g)
%inibition(
11l
EC50 (µg/m L) >1000
155 ± 42
13 ± 3c
42 ± 1b
49d
499
306 ± 70
24 ± 5b
EtOH
27.5 ± 0.4cd
48e
511
69 ± 5
6.6 ± 0.3de
Hx
22 ± 2efg
13k
>1000
130 ± 20
11 ± 1cd
EtOAc
55 ± 6a
55c
441
430 ± 21
33 ± 3b
EtOH
28 ± 1cd
75b
309
106 ± 8
9.3± 0.6cde
150 bar/40 °C 150 bar/50 °C 150 bar/60 °C 200 bar/40 °C 200 bar/50 °C 200 bar/60 °C 300 bar/40 °C 300 bar/50 °C 300 bar/60 °C 150 bar/50°C
16 ± 2h
14j
>1000
41 ± 5
4.6 ± 0.3de
19.8 ± 0.4fgh
6n
>1000
216 ± 11
16 ± 2c
15.2 ± 0.5h
16i
>1000
52 ± 5
6 ± 1de
25 ± 2def
10m
>1000
95 ± 9
8.6 ± 0.6cde
21 ± 1efgh
14j
>1000
105 ± 10
13 ± 6c
26 ± 1cde
14j
>1000
23 ± 8
3.3 ± 0.5e
20 ± 1efgh
26g
>1000
157 ± 20
13 ± 1c
15 ± 2h
16i
>1000
186 ± 11
15 ± 1c
21.5 ± 0.5efg
26g
>1000
40 ± 8
4 ± 1de
18 ± 2gh
13k
>1000
57 ± 10
6 ± 1de
150 bar/50°C
46 ± 3b
34f
713
96 ± 9
9 ± 1cde
150 bar/50°C
24 ± 3def
25h
948
112 ± 4
9.8 ± 0.3de
100 bar/40 °C -
-
53c
339
-
-
268a ± 13
89.7a
-
-
93.1 ± 0.2
TPC(1,5)(mgGA E/g extract)
%AA(2,
30.3 ± 0.3c
EtOAc
(2)
5)
(3)
4,5)
Total phenolic content; Antioxidant activity evaluated by free radical scavenging activity (DPPH·); (3) Effective concentration at 50%; (4) Antioxidant activity evaluated by ABTS method; (5) Same letters at the same column indicated no significant difference at level of 5 % (p < 0.05); (6) Benelli et al. [17]. Hx, hexane; EtOAc, ethyl acetate; EtOH, ethanol. UAE, ultrasound extraction; SOX, soxhlet; SFE-CO2, supercritical fluid extraction with CO2.
Table 3. Relative composition profile, in % peak area, of black pepper extracts. tr (m in)
16 .9 68
18 .4 73
Ident
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1: UAE Hex; 2: UAE EtOAc; 3: UAE EtOH; 4: SOX Hex; 5: SOX EtOAc; 6: SOX EtOH; 7: SFE 150 bar/40 °C; 8: SFE 150 bar/50 °C; 9: SFE 150 bar/60 °C; 10: SFE 200 bar/40 °C; 11: SFE 200 bar/50 °C; 12: SFE 200 bar/60 °C; 13: SFE 300 bar/40 °C: 14: SFE 300 bar/50 °C; 15: SFE 300 bar/60 °C; 16: SFE 150 bar/50 °C + 2,5 % EtOH; 17: SFE 150 bar/50 °C + 5 % EtOH; 18: SFE 150 bar/50 °C + 7,5 % EtOH; 19: PLE EtOH. tr: retention time.
S0896-8446(17)30181-X http://dx.doi.org/doi:10.1016/j.supflu.2017.05.001 SUPFLU 3912
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
13-3-2017 26-4-2017 1-5-2017
Please cite this article as: K´atia S.Andrade, Guilherme Trivellin, Sandra R.S.Ferreira, PIPERINE-RICH EXTRACTS OBTAINED BY HIGH PRESSURE METHODS, The Journal of Supercritical Fluidshttp://dx.doi.org/10.1016/j.supflu.2017.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PIPERINE-RICH EXTRACTS OBTAINED BY HIGH PRESSURE METHODS
Kátia S. Andrade1, Guilherme Trivellin1, Sandra R. S. Ferreira1*. 1
Chemical and Food Engineering Department – Federal University of Santa Catarina – EQA/UFSC – C.P. 476, CEP 88040-900, Florianópolis, SC, Brazil.
*
Corresponding author: [email protected] (S.R.S. Ferreira).
GRAPHICAL ABSTRACT
HIGHLIGHTS
Black pepper extraction with ethanol provides a product with high antioxidant potential;
SFE is suitable technology to provide extracts with high quality in terms of chemical composition and solvent free;
PLE, SFE and LPE extracts of black pepper have piperine and caryophyllene as major compounds.
PLE technique is a promising method to obtain bioactive extracts from black pepper.
ABSTRACT
Chemical composition and antioxidant activity of black pepper extracts were studied. The high pressure methods Supercritical Fluid Extraction (SFE) (at 40, 50 and 60 °C and from 150 to 300 bar) and pressurized liquid extraction (PLE) with ethanol were compared to Soxhlet (SOX) and ultrasound-assisted extraction (UAE). The comparison considered process yield, extract composition and antioxidant activity, evaluated by DPPH (1,1diphenyl-2-picrylhydrazil) and ABTS [2,2-azino-bis (3-ethylbenzotiazoline-6-sulphonic acid)] methods, and total phenolic content by Folin-Ciocalteau procedure. Highest global yield (X0 = 10.3 ± 0.6 %) and antioxidant potential was obtained by SOX extract with ethanol. The chemical profile was evaluated by CG-MS analysis. The main compound identified was piperine for the supercritical and ethanolic extracts. Despite the several researches concerning SFE of black pepper, the importance of this raw material justifies a broader understanding of this material, added to the novelty of PLE and ABTS procedures applied for black pepper material.
Keywords: Supercritical technology; piper nigrum; piperine, antioxidant activity.
1. Introduction The black pepper plant is the most commonly used spice in the world due to its commercial, economical, nutritional, and medicinal value. The seeds are commonly used to enhance the taste and flavor of food, while the essential oil plays a significant role in food processing, health, and nutrition by preventing or delaying spoilage of foods due to its antioxidant and antimicrobial properties [1].
Brazil is among the three largest producers of peppers in the world and has about 90 % of its production exported [2]. The components of the pepper’s extract that contribute to its value as a food additive are the volatile oil for its aroma and the alkaloid compounds for the pungency. Black pepper is used in skin care, muscle and joint pains, and in improving blood circulation and respiratory systems [3]. Piperine, the major component present in black pepper, is an alkaloid responsible for pungency and irritation and it has demonstrated antioxidant and antimicrobial properties. This alkaloid is an alternative and complementary treatment to various disorders [4], has pharmacological impact on nervous and neuromuscular systems, and exercises sedative effect and helps in digestion [3]. Ecofriendly techniques of extraction and recovery of bioactive compounds are very attractive in the last years. Among these green technologies, the extraction assisted by ultrasound, by pressurized liquid and extraction using supercritical fluids must be highlighted [5–7]. Ultrasound-assisted extraction (UAE) is based on the use of the energy of sound waves that provoke expansion and compression cycles in a medium (mechanical vibration). This phenomena accelerates the extraction of compounds in reduced time due to the influence on the mass transport, solvent/sample ratio and temperature [8]. Pressurized liquid extraction (PLE) consists of the continuous solvent flow through a fixed solid bed of particles placed inside the extraction column. The main advantages of PLE are higher extraction efficiencies requiring lower amounts of solvents, reduced extraction time and the use of solvents classified as Generally Recognized as Safe (GRAS) [9–11]. Supercritical fluid extraction (SFE) is a green process and gained attention over traditional methods because it protects against thermal degradation and solvent contamination of the extracts [12].
Therefore, this study proposes to obtain natural extracts from black pepper (piper nigrum) in order to evaluate the application of green technologies in the recovery of compounds of high added value, by analyzing the composition profile and the antioxidant activity of the extracts. Black pepper, as the most important spice commodity, presents particular characteristics that always raise academic interest, either concerning the crop or the processing differences. Also, to the bests of our knowledge and to broad the use of green methods to obtain black pepper extracts, no PLE research was detected. Therefore, although there are several researches concerning SFE of black pepper, the importance of this raw material justifies a broader understanding of this material, together with the innovation regarding the PLE and ABTS procedures applied for black pepper material.
2. Materials and methods
2.1 Raw material and sample preparation The unripe fruits of Piper nigrum L. cultivar Bragantina was gently supplied by a Farm located at São Mateus City (Espírito Santo, Brazil) (18° 43’ 37” S; 40° 05’ 51” W; 26 m of altitude). The plant was identified and a voucher specimen (number 15422; 11/18/2014) was deposited at Municipal Herbarium collection (São Paulo, Brazil). The raw material provided presented 15 ± 1 % (w/w) of moisture and volatile content, determined according to the 950.46B method of AOAC [13]. The black pepper samples were stored at room temperature (± 25 °C) and used as supplied, avoiding a previous drying step that could cause degradation in the raw material. The pepper grains were ground in a knife mill and characterized by size classification in a vertical vibratory sieve shaker. The mean particle diameter, calculated by mean size distribution as described by Gomide [14], indicated a mean value of 570 ± 1 µm. The grounded material was stored
at – 18 °C until the extractions were performed. All solvents used in the experiments were of analytical grade (PA) purchased from Lafan Química Fina LTDA. Carbon dioxide (99.9 % purity) was purchased from White Martins S/A (São Paulo, Brazil) and delivered at pressure up to 60 bar.
2.2 Extraction methods
2.2.1 Supercritical fluid extraction (SFE) A high-pressure unit, previously described by Zetzel et al. [15], was used for the SFE with CO2 by following the extraction procedure from Michielin et al. [16] to obtain the black pepper extracts. A co-solvent pump was connected to the extraction line in order to supply the modifier (organic solvent at high-pressure). Briefly, the extraction consisted of placing a fixed mass of 20 g of the dried raw material (grinded black pepper) inside the extractor cell to form the fixed bed of particles, followed by the control of the process variables (temperature and pressure). The extraction was then performed and the solute collected in amber flasks and weighed on an analytical balance (OHAUS, Model AS200S, NJ, USA). The SFE with CO2 was conducted at temperatures of 40, 50 and 60 °C and pressures of 150, 200 and 300 bar, at constant solvent flow rate of 8 ± 2 g min-1. The extraction time was set at 4 h according to the kinetic extraction curve, performed at 200 bar, 50 °C (intermediary conditions of extraction) and 8 ± 2 g CO2 min-1. At the end of this period, most of the extractable solute mass had been recovered. The solvent density values were obtained according to Angus et al. [17]. The co-solvent (CS) assays were performed at 150 bar and 50 °C at constant solvent flow rate of 8 ± 2 g min-1. Ethanol (EtOH) was used as modifier in concentrations of 2.5, 5.0 and 7.5 % (w/w) related to the CO2 mass. The co-solvent was separated from the extract according to the procedure
described for the low pressure methods (Section 2.2.2). The results were expressed in terms of extraction global yield (X0).
2.2.2 Low pressure extractions (LPE) The low pressure extraction methods, Soxhlet (SOX) and ultrasound-assisted (UAE), were applied using three different solvents: hexane (Hx), ethyl acetate (EtOAc) and ethanol (EtOH), with polarity index of 0, 4.4 and 5.2, respectively [18]. The SOX extraction was performed according to 920.39C method of AOAC [13]. Briefly, 150 mL of solvent recycling over 5 g of dried sample, in a Soxhlet apparatus for 6 h extraction conducted at the solvent boiling temperature. The ultrasound-assisted extractions consisted of placing 7 g of raw material and 210 mL of solvent inside a covered glass balloon. The extraction was performed at room temperature during 45 min. The equipment used was an ultrasonic cleaner bath (USC700/55 kHz, Unique Ltda., Indaiatuba/SP, Brazil), which operates in a frequency of 55 kHz and potency of 100 W [19]. The residual solvent from all extracts (SOX and UE with three solvents) was eliminated in a rotatory evaporator (Fisatom, 802, Brazil), supplied with cooling and vacuum control. The evaporation temperature was adjusted to a level below the solvent boiling point in order to avoid thermal degradation of the extracts. The results, expressed in extraction global yield (X0), represent the mean values ± standard deviation from duplicate experiments, for each LPE assay.
2.3 Pressurized liquid extraction (PLE) The dynamic extraction method was used in PLE with ethanol as solvent and it was carried out according to Viganó et al. [20]. This method consists of the continuous solvent
flow through a fixed solid bed of particles placed inside the extraction column. Approximately 20 g of ground black pepper was loaded in the extraction vessel, forming a fixed bed inside a stainless steel column. Then, ethanol was pressurized using a HPLC pump (Waters, EUA) until 100 bar and passed through a coil in a heating bath (Microquímica, Brazil) to reach the process temperature (40 °C). The ethanol flow rate was kept constant at 3 mL min-1 and the extraction time was set in 60 min. The extracts were collected in glass flasks, the solvent was removed following the procedure described in Section 2.2.2 and then, the dried extract was stored at -18 °C until the analysis were performed. The experiments were carried out in duplicate and global yield was expressed by mean ± standard deviation. These assays were performed preliminarily to evaluate the PLE feasibility to obtain extracts with antioxidant potential, although, complementary studies (ongoing research) may provide insights related to the influence of the PLE operational conditions and solvent types in extract quality.
2.4 Determination of total phenolic content (TPC) The TPC was determined according to the Folin-Ciocalteu spectrophotometric method [21]. Briefly, the reaction mixture was composed by 0.1 mL of extract (concentration of 1667 mg/L), 7.9 mL of distilled water, 0.5 mL of Folin–Ciocalteu reagent (a mixture of phosphomolybdate and phosphotungstate) and 1.5 mL of 20 % sodium carbonate, placed in opaque flasks. The flasks were agitated and allowed to stand for 2 h, and the absorbance was measured at 765 nm in a spectrophotometer (Femto, 800XI, Brazil). The TPC was calculated according to a standard curve (y= 0.0011x + 0.0352; R²=0.9951), previously prepared with gallic acid as standard (galic acid equivalent: GAE). The analysis was performed in triplicate and the results expressed as mg GAE g-1 extract as mean ± standard deviation.
2.5 Antioxidant potential The antioxidant potential of the black pepper extracts, obtained by different extraction methods (SFE, SFE-CS, SOX and UAE), was evaluated by two scavenging activity procedures, DPPH· and ABTS methods. The results were compared with the antioxidant activity from the synthetic compound BHT (butylated hydroxytoluene). The antioxidant potential of the PLE extract was only evaluated by DPPH· method.
2.5.1 Free radical scavenging activity (DPPH·) The free radical scavenging of black pepper extracts was evaluated using 1,1diphenyl-2-picrylhydrazil (DPPH·) as described by Mensor et al. [22]. Briefly, each extract was mixed with a 0.3 mM DPPH· ethanolic solution to give final concentrations of 5, 10, 25, 50, 125, 250 and 500 µg extract mL-1 DPPH· solutions. After 30 min at room temperature, the absorbance values were measured at 517 nm and converted into percentage of antioxidant activity (% AA). This activity was also presented as the effective concentration at 50 % (EC50), i.e., the concentration of the test solution required to give 50 % decrease in the absorbance of the test compared to that of a blank solution, and expressed in µg of extract/mL DPPH·. The EC50 values were calculated from the linear regression of the % AA curves obtained for all extract concentrations. The % AA and EC50 for all extracts were obtained considering the mean value of triplicate assays. This method was also used to describe the antioxidant potential of extracts obtained by PLE method.
2.5.2 ABTS radical scavenging assay
The ABTS [2,2-azino-bis (3-ethylbenzotiazoline-6-sulphonic acid)] assay was carried out according to Re et al. [23] and the ABTS activity was calculated considering the standard concentration curve. The synthetic vitamin E, Trolox (6-hidroxy-2,5,7,8tetramethylchroman- 2-carboxylic acid) was used as antioxidant reference, which was prepared in ethanol and stored as standard solution. Briefly, the ABTS was dissolved in water up to a concentration of 7.0 mM, and subjected to reaction with potassium persulphate (2.45 mM) for the radical formation. The ABTS solution was diluted with ethanol to absorbance of 0.70 ± 0.05 at 754 nm. Solutions were prepared with 20 μL of the extract at concentrations of 100, 250, 500 and 1000 mg mL-1, in triplicate. Than 980 μL of ABTS solution was added to the test tubes and after 6 minutes the absorbance was read at spectrophotometer (Femto, 800XI, Brazil). Results were expressed as MTEAC/g (Trolox Equivalent Antioxidant Capacity per g extract), such as mean ± standard deviation.
2.6 Chemical profile The identification and relative quantification of the volatiles compounds present in the black pepper extracts were performed by a gas chromatograph equipped with a mass spectrophotometer (GC/MS, model 7890 A, mass detector 5975C, Agilent Technologies, USA), attached to a HP-5MS column (30 m x 0.25 mm internal diameter x 0.25 µm film thickness, Agilent Technologies, USA). The carrier gas was helium with flow rate of 1 mL min-1, split ratio of 1:50, injector temperature of 250 °C and Thermal Aux 2 (MSD Transfer Line) 250 °C, while column temperature programmed from 70 to 280 °C at a rate of 3 °C min-1. The main components of black pepper extracts were identified by comparing the mass spectra and the retention time with NIST 11 Mass Spectral Library available on equipment.
2.7 Statistical analysis The global yield (X0), TPC and the potential antioxidant results were evaluated statistically by software SAS for Windows version 6.0, at 5 % level of significance, in order to identify significant differences between values of global yield, as a function of temperature, pressure, solvent/extraction type, and percentage of antioxidant activity. All analyses were carried out in triplicate and the results were expressed as means ± standard deviation (SD).
3 Results and discussion
3.1 Global yield (X0) of LPE, SFE and PLE The global yield (X0), defined as the amount of solute extractable by the solvent at the extraction conditions, indicates, quantitatively, the process efficiency. The yield results obtained for the different extraction methods and solvents (Soxhlet, UAE, SFE with CO2 and PLE) for black pepper are presented in Table 1.
Table 1. Global yield (X0) of black pepper extracts obtained by low pressure extractions (LPE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE).
The results presented in Table 1 indicate that the best yield was obtained by the Soxhlet extraction using ethanol as solvent. It is possible to observe a yield increase trend due to the increase in solvent polarity, suggesting that compounds present in plant matrix have intermediate to high polarity. Moreover, the dielectric constant may explain the superior performance of the extraction with ethanol compared to other solvents. The
dielectric constant is the ability to attenuate electric fields and thus assist in the interaction between charges. Thus, as this property has a value significantly higher for the ethanol, the solvent molecules are strongly linked to the polar components contributing to the increased extraction yield [24]. Comparing the low pressure methods for the same solvent, it was observed that the Soxhlet extraction had higher yields compared to ultrasound-assisted leaching extraction. The operating temperature of the recycle solvent and the interactions between solvent and plant matrix, characteristic of Soxhlet extraction, may contribute to increase the solubility of compounds of different types, raising the extraction yield [25–27]. The global yield of PLE with ethanol is comparable to the ultrasound-assisted extraction using ethanol and ethyl acetate as solvents. Moreover, it is important to note that in the PLE, despite the similar extraction time between the methods, the PLE advantages are lower solvent volume and no filtration step needed. In this work, only one temperature condition was evaluated, however, the use of higher temperatures may improve the solubility of the compounds of interest and their diffusivity, a subject under investigation at the ongoing research about PLE used to obtain black pepper extract. The extract to solvent ratio (E/S) represents the effectiveness of the extraction method or conditions of pressure and temperature, for each extraction method. Therefore, higher E/S ratio represents higher extraction method efficiency because less solvent is required to obtain a given mass of extract. From the E/S values presented in Table 1 it is possible to observe the best performance of the procedure PLE with ethanol (5.88 mg mL1
), compared to ethanolic extracts from other methods, for instance, SOX provided E/S
of 3.57 mg mL-1 and UAE of 1.89 mg mL-1.
A broader comparison of the extraction efficiency among the different methods using ethanol as solvent is observed in Figure 2, which relates the extraction yield, the E/S ratio and the extraction time for the methods PLE, UAE and SOX.
Figure 2. Extraction evaluation of PLE, SOX and UAE using ethanol as solvent.
The SOX yield reached the highest value (10.3 %), compared to other methods, probably caused by the solvent recycling, which leads to the raw material exhaustion with time, reducing the extraction efficiency (E/S). On the other hand, PLE and UAE methods present process time and yield values very similar, however the PLE method provides higher process efficiency (high E/S ratio), compared to UAE and SOX. Therefore, taking into account the extraction time, the volume of solvent used and the mass of extract obtained, it is possible to suggest that the PLE method was more effective that SOX and UAE, to obtain black pepper extracts. SFE with pure CO2 at the highest condition (300 bar and 60 °C) produced yield values very close to the ones obtained by Soxhlet and ultrasound-assisted extraction with hexane, suggesting the extraction of compounds with lipophylic characteristic, more easily dissolved in nonpolar solvents. The poor performance from the non-polar solvents (CO2 and hexane) is attributed to the low fatty acids content in the black pepper ground. In the extraction with supercritical CO2, the evaluation of global yield extraction, obtained under different conditions of temperature and pressure, indicates the effect of solubility of the solvent and consequently the influence on the process yield. According to Table 1, the highest yield, in supercritical conditions, for the black pepper extracts was 3.2 ± 0.3 %, obtained for the condition of 300 bar and 60 °C. This value shows no significant difference, at level of 5 %, to the results obtained at 300 bar
and 50 °C. The results achieved by the other extraction conditions also show no significant difference among each other. The pressure increase at constant temperature enhances the extraction yield, due to the increase in the CO2 density and consequently in the solvent power [12]. Besides, the increased pressure can lead to disruptions in the plant cells, facilitating the release of compounds that were not previously available, and thus increasing the yield of the process, as reported in the literature [27,28]. The lowest yields were obtained at 150 bar, at all temperatures. The effect of temperature on extraction yield, at constant pressure, occurs by two mechanisms: the increase in process temperature increases the solubility due to increased vapor pressure of the solute and on the other hand reduces the solubility due to the decrease in density the solvent. This behavior occurs near the critical point, where small changes in temperature and/or pressure are accompanied by large variations in solvent density [12,28]. Comparing the yields obtained by supercritical CO2 with the results obtained by conventional extraction (SOX and UAE) and PLE, it is observed that the low pressure extractions and PLE using ethanol as a solvent produced higher yield values to those achieved by SFE. These results can be explained by the extraction of more polar compounds, not soluble in CO2, a nonpolar solvent. Additionally, for the Soxhlet extractions, the solvent recycling, the extended extraction time and the high amount of solvent aid the yield enhancement, compared to other methods [29,30].
3.1.1 SFE with CO2 and co-solvent (CS) In order to improve the efficiency of SFE technique by increasing the recovery of polar compounds, EtOH, a green modifier, was selected as a co-solvent. The co-solvent
and the extraction conditions were selected based on the global yield, TPC and antioxidant activity results obtained by low pressure extractions and SFE with CO2. Consequently, the extraction was performed at 150 bar and 50 °C using ethanol concentrations of 2.5, 5.0 and 7.5 % (w/w). The results obtained by the SFE with cosolvent for black pepper are presented in Figure 2.
Figure 2. Global yield (X0) of black pepper extracts obtained by SFE - CO2 with cosolvent.
The SFE with ethanol as co-solvent increased the yield values for all ethanol concentrations and conditions tested. This behavior is explained by the increase in the solubility of polar compounds in the mixture ethanol/CO2, compared to the solubility in pure CO2. Furthermore, not only the solubility of a certain component increases with the use of co-solvent, but also the number of components solubilized by the solvent, which reduces the selectivity and increases the process yield [31,32]. A larger amount of co-solvent favors solute/solvent interactions and increases the yield. Generally, the type of co-solvent has a greater impact on the extraction efficiency than its concentration, because of the type of matrix-compounds interaction and physicochemical parameters of the co-solvent. Normally, better results are achieved at higher concentrations, but in the some cases the behavior is unlike [33].
3.2 Total phenolic content (TPC) and antioxidant activity
The results for the total phenolic content and antioxidant activity are presented in Table 2, obtained for all extract samples analyzed, and compared to the results presented
by the synthetic product BHT, as standard sample. The antioxidant potential was evaluated by two scavenging methods with the radicals DPPH· and ABTS. Both radical scavenging methods used in this work are popular for determination of the antioxidant capacity of foodstuff due to the simple, rapid, sensitive, and reproducible procedures. The extracts were obtained from black pepper using different extraction methods (LPE, SFE, PLE).
Table 2. Antioxidant activity and TPC of black pepper extracts.
The values of total phenolic content obtained by SFE show no variation behavior with changes in pressure or temperature. Most of supercritical extract showed similar results and lower to that found in commercial antioxidant, BHT. The highest data for SFE was obtained at 150 bar and 50 °C using 5 % ethanol as co-solvent (46 ± 1 mgGAE/ gextract). This behavior can be explained by the increase in the solubility of polar compounds in the mixture ethanol + CO2, compared to the solubility in pure CO2 [32]. The addition of ethanol as a co-solvent in the SFE of black pepper favors the extraction of phenolic compounds, except for the 2.5 % concentration of co-solvent, when the result was statistically equal to extraction with pure CO2. Andrade et al. [26] also observed a reduction in the total phenolic content in coffee grounds extracts with increased concentration of ethanol in SFE. According Campos et al. [28], there is an optimum concentration of co-solvent to be used in the SFE to achieve better yields and better antioxidant activity. Above this concentration, which in this study is 5 %, there was a reduction in the extraction of phenolic compounds, indicative of antioxidant activity. For the LPE methods, the best results of TPC were obtained with ethyl acetate as solvent, for the Soxhlet and UAE method, reaching 55 ± 6 mgGAE/gextract and 42 ± 1
mgGAE/gextract, respectively. The SFE with ethanol (5 %) produced TPC of 46 ± 3 mgGAE/gextract, statistically equal to that obtained in the ultrasound-assisted extraction using ethyl acetate as solvent. The black pepper extracts that showed the higher values of antioxidant activity from the DPPH method were obtained by Soxhlet using ethanol (75 %) and ethyl acetate (55 %) as solvents, respectively. This result may be compared to TPC values, which indicate that its antioxidant activity may be not directly related to its phenolic content. The extract obtained by PLE showed 55 % of antioxidant activity determined by DPPH method, which suggest that PLE is a promising technique to obtain bioactive extracts. Also the increase in extraction temperature may lead to the recovery of new antioxidant compounds formed from Maillard, caramelization and thermo oxidation reactions [34] . The EC50 results, calculated from DPPH data for the supercritical extracts were above the 250 µg mL-1. Bellow this value the extract is considered a good antioxidant product [28]. The behavior of the SFE may be associated to the low amount of phenolic compounds with intermediate to high polarity, since CO2, a non-polar solvent, does not favor the solubilization of such components. Similarly to the TPC results, the addition of 5% co-solvent increased the antioxidant activity of extract compared to that obtained by CO2, due to the higher polarity of the solvent mixture (CO2 and ethanol) compared to the supercritical CO2, favoring the extraction of phenolic compounds [7]. The results from Table 2 also show that Soxhlet and UAE extraction with ethyl acetate presented the highest AA % behavior by the ABTS method, with values of 430 ± 21 µMTEAC/gextract and 306 ± 70 µMTEAC/gextract, respectively. The best antioxidant capacities for supercritical extracts were found for pressure of 150 bar and 50 °C, probably because the components responsible for antioxidant characteristics detected by
the ABTS method were present in higher concentrations. This situation suggests that the presence of ethanol affect the solubilization of compounds responsible for the antioxidant activity of the extracts, detected by this method of analysis. No earlier studies are reported in the literature regarding the ABTS assay of black pepper extract obtained by the SFECO2. Natural extracts may be more beneficial than isolated bioactive compounds, because the synergic interaction of compounds may enhance the properties of the individual components. Moreover, the use of natural extracts may be beneficial considering that maximum lawful levels for synthetic food additives are established based on various toxicological parameters which are usually not applicable to naturally occurring compounds [35,36].
3.3 Chemical profile The gas chromatography analysis was performed for the black pepper extracts in order to evaluate the volatile fraction, mainly obtained by low polar solvents, such as supercritical CO2 or hexane. The chemical profile results are presented in Table 3, with the compounds identified and the relative composition (integrated composition) for all extracts for black pepper obtained by supercritical fluid extraction (SFE), low pressure extraction (SOX and UAE) and pressurized liquid extraction (PLE). The major compounds identified in terms of area percentage relative and/or impact in different extracts were piperine and caryophyllene.
Table 3. Relative composition profile, in % peak area, of black pepper extracts.
Piperine is the alkaloid responsible for the pungency of black pepper and it has been used in many forms in traditional medicine. Several biological activities are associated with piperine, such as antioxidant, antitumor, antidepressant activity, among others [37– 39]. No trends were detected concerning the effects of pressure and temperature on composition of supercritical extracts. The identified compounds exhibit valuable biological activity, which may suggest the use of extracts in various therapeutic applications, considering, of course, the synergistic effect between the compounds. A similar composition was found by Bagheri et al. [40] and Ferreira et al. [41] for black pepper essential oil obtained by SFE at 300 and 150 bar, 40 °C, respectively. In that case, the majoritary compounds were sabinene and caryophyllene. Among the identified components, it is important to highlight the sesquiterpenes with reports of antitumor activities, such as caryophyllene [42] and humulene [43]. The extracts obtained by low polar solvents provided a wider varied of sesquiterpenes, compared to other extracts. Based on the results of extract composition and yield, two extracts obtained by SFE (200 and 300 bar, 40 °C) were submitted to the analysis of antitumor activity in the work developed by Grinevicius [44]. In vivo treatment with the black pepper SFE of isogenic Balb-c mice bearing Ehrlich ascites carcinoma showed higher inhibition and increased the survival time compared to ethanolic extract. The highest concentration of piperine and sesquiterpenes in these extracts may have caused the cytotoxicity and the inhibition of the cell proliferation through apoptosis, resulting in best antitumor performance of the supercritical extract. Although the piperine was the major compound detected in the black pepper extracts, the Piper nigrum biological activities may be a result of the synergic interaction among the different compounds present in the raw material. Thus, further
studies related to black pepper extracts may be of interest, starting from this work that target the piperine component.
4. Conclusions The use of black pepper as raw material for different extraction methods is promising due to the high quality of the substances present in this vegetable matrix. When comparing the different extraction methods, besides the determination of the process yield, it is also necessary to quantify the antioxidant potential of the product (extract) by diverse procedures and also evaluate the chemical composition of the extracts. SOX and UAE presented highest extraction yield when using ethanol as solvent. SFE extracts presented lowers yields when compared to SOX and UAE methods. The use of ethanol as co-solvent in SFE increased the yield extraction and also the antioxidant activity when 5 % of ethanol was added to the process. PLE is an emerging technology that has shown good results in relation to the extraction of bioactive compounds and need further studies in order to evaluate the influence of the different operational conditions on the extracts quality. The supercritical fluid extraction is an effective technique for the recovery of extracts rich in piperine, comparable to extraction by Soxhlet with ethanol, providing a high valueadded and solvent-free extract. Finally, it is important to point that each extraction procedure presents particular characteristics to provide products for different applications. The challenge is to explore the extraction in order to optimize the extract composition profile, related to the raw material specificities.
Acknowledgments The authors wish to acknowledge CAPES (Coordination for the Improvement of Higher Education Personnel) for the scholarship, CNPq (National Council of
Technological and Scientific Development) (Process number 473153/2012-2) for the financial support.
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FIGURE CAPTIONS
Figure 1. Extraction evaluation of PLE, SOX and UAE using ethanol as solvent.
Figure 2. Global yield (X0) of black pepper extracts obtained by SFE - CO2 with cosolvent (ethanol) at 150 bar and 50 °C.
FIGURE GRAPHICS
12
10
8
6
4
2
0 PLE
UAE X0 (%)
E/S (mg/mL)
SOX Extraction time (h)
Figure 1. Extraction evaluation of PLE, SOX and UAE using ethanol as solvent.
6 5
Yield (%)
4 3 2 1 0 0
5 2.5 Co-solvent (%, w/w)
7.5
Figure 2. Global yield (X0) of black pepper extracts obtained by SFE - CO2 with cosolvent (ethanol) at 150 bar and 50 °C.
TABLES Table 1. Global yield (X0) of black pepper extracts obtained by low pressure extractions (LPE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE). Extraction Method
40 °C
SFE-CO2
50 °C
60 °C
LPE
SOX
UAE
PLE 100 bar/40 °C (1)
ρCO2(1) (g/cm³) 0.781
1.5 ± 0.4de
E/S(4) (mg/mL) 0.12
200 bar
0.840
2.1 ± 0.1cde
0.24
300 bar
0.911
2.4 ± 0.4bc
0.22
150 bar
0.701
1.28 ± 0.01e
0.10
200 bar
0.785
1.6± 0.4cde
0.14
300 bar
0.871
2.7 ± 0.1ab
0.26
150 bar
0.606
1.4 ± 0.3e
0.08
200 bar
0.724
2.2 ± 0.2bcd
0.17
300 bar
0.830
3.2 ± 0.3a
0.28
Process Parameters 150 bar
X0 (%)(2)
Solvents
SPI(3)
Hx
0
4.1 ± 0.5C
1.35
EtOAc
4.3
6.5 ± 0.3B
2.50
EtOH
5.2
10.3 ± 0.6A
3.57
Hx
0
3.1 ± 0.1E
1.04
EtOAc
4.3
5.6 ± 0.1C
1.72
EtOH
5.2
5.3 ± 0.5C
1.89
Solvents EtOH
SPI(3) 5.2
5.265 ± 0.007C
5.88
CO2 density (Angus, Armstrong & Reuck [17]). Same letters indicated no significant difference at level of 5% (p < 0.05). (3) Solvent polarity index (4) Extract to solvent ratio Hx, hexane; EtOAc, ethyl acetate; EtOH, ethanol. (2)
Table 2. Antioxidant activity and total phenolic content for black pepper extracts Extracti on Methods UAE
SOX
SFECO2
SFECO2+2.5 % EtOH SFECO2+ 5 % EtOH SFECO2+ 7.5 % EtOH PLE EtOH BHT(6) (1)
Process Paramete rs Hx
TEAC(4)(μMTEA C/g)
%inibition(
11l
EC50 (µg/m L) >1000
155 ± 42
13 ± 3c
42 ± 1b
49d
499
306 ± 70
24 ± 5b
EtOH
27.5 ± 0.4cd
48e
511
69 ± 5
6.6 ± 0.3de
Hx
22 ± 2efg
13k
>1000
130 ± 20
11 ± 1cd
EtOAc
55 ± 6a
55c
441
430 ± 21
33 ± 3b
EtOH
28 ± 1cd
75b
309
106 ± 8
9.3± 0.6cde
150 bar/40 °C 150 bar/50 °C 150 bar/60 °C 200 bar/40 °C 200 bar/50 °C 200 bar/60 °C 300 bar/40 °C 300 bar/50 °C 300 bar/60 °C 150 bar/50°C
16 ± 2h
14j
>1000
41 ± 5
4.6 ± 0.3de
19.8 ± 0.4fgh
6n
>1000
216 ± 11
16 ± 2c
15.2 ± 0.5h
16i
>1000
52 ± 5
6 ± 1de
25 ± 2def
10m
>1000
95 ± 9
8.6 ± 0.6cde
21 ± 1efgh
14j
>1000
105 ± 10
13 ± 6c
26 ± 1cde
14j
>1000
23 ± 8
3.3 ± 0.5e
20 ± 1efgh
26g
>1000
157 ± 20
13 ± 1c
15 ± 2h
16i
>1000
186 ± 11
15 ± 1c
21.5 ± 0.5efg
26g
>1000
40 ± 8
4 ± 1de
18 ± 2gh
13k
>1000
57 ± 10
6 ± 1de
150 bar/50°C
46 ± 3b
34f
713
96 ± 9
9 ± 1cde
150 bar/50°C
24 ± 3def
25h
948
112 ± 4
9.8 ± 0.3de
100 bar/40 °C -
-
53c
339
-
-
268a ± 13
89.7a
-
-
93.1 ± 0.2
TPC(1,5)(mgGA E/g extract)
%AA(2,
30.3 ± 0.3c
EtOAc
(2)
5)
(3)
4,5)
Total phenolic content; Antioxidant activity evaluated by free radical scavenging activity (DPPH·); (3) Effective concentration at 50%; (4) Antioxidant activity evaluated by ABTS method; (5) Same letters at the same column indicated no significant difference at level of 5 % (p < 0.05); (6) Benelli et al. [17]. Hx, hexane; EtOAc, ethyl acetate; EtOH, ethanol. UAE, ultrasound extraction; SOX, soxhlet; SFE-CO2, supercritical fluid extraction with CO2.
Table 3. Relative composition profile, in % peak area, of black pepper extracts. tr (m in)
16 .9 68
18 .4 73
Ident
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1: UAE Hex; 2: UAE EtOAc; 3: UAE EtOH; 4: SOX Hex; 5: SOX EtOAc; 6: SOX EtOH; 7: SFE 150 bar/40 °C; 8: SFE 150 bar/50 °C; 9: SFE 150 bar/60 °C; 10: SFE 200 bar/40 °C; 11: SFE 200 bar/50 °C; 12: SFE 200 bar/60 °C; 13: SFE 300 bar/40 °C: 14: SFE 300 bar/50 °C; 15: SFE 300 bar/60 °C; 16: SFE 150 bar/50 °C + 2,5 % EtOH; 17: SFE 150 bar/50 °C + 5 % EtOH; 18: SFE 150 bar/50 °C + 7,5 % EtOH; 19: PLE EtOH. tr: retention time.