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Application of conventional and non-conventional extraction approaches for extraction of Erica carnea L.: Chemical profile and biological activity of obtained extracts
Accepted Manuscript Title: Application of conventional and non-conventional extraction approaches for extraction of Erica carnea L.: chemical profile and biological activity of obtained extracts Authors: Vesna Veliˇckovi´c, Saˇsa Ðurovi´c, Marija Radojkovi´c, ˇ Aleksandra Cvetanovi´c, Jaroslava Svarc-Gaji´ c, Jelena Vuji´c, Sre´cko Trifunovi´c, Pavle Z. Maˇskovi´c PII: DOI: Reference:
S0896-8446(17)30033-5 http://dx.doi.org/doi:10.1016/j.supflu.2017.03.023 SUPFLU 3892
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
12-1-2017 25-3-2017 27-3-2017
Please cite this article as: Vesna Veliˇckovi´c, Saˇsa Ðurovi´c, Marija Radojkovi´c, ˇ Aleksandra Cvetanovi´c, Jaroslava Svarc-Gaji´ c, Jelena Vuji´c, Sre´cko Trifunovi´c, Pavle Z.Maˇskovi´c, Application of conventional and non-conventional extraction approaches for extraction of Erica carnea L.: chemical profile and biological activity of obtained extracts, The Journal of Supercritical Fluidshttp://dx.doi.org/10.1016/j.supflu.2017.03.023 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.
Application of conventional and non-conventional extraction approaches for extraction of Erica carnea L.: chemical profile and biological activity of obtained extracts
Vesna Veliţkoviš1, Saša Đuroviš2**, Marija Radojkoviš2, Aleksandra Cvetanoviš2, Jaroslava Švarc-Gajiš2, Jelena Vujiš1, Sreško Trifunoviš1, Pavle Z. Maškoviš3*
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University of Kragujevac, Faculty of Science, Department of Chemistry, Radoja Domanoviša 12, 34000 Kragujevac, Serbia
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University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
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University of Kragujevac, Faculty of Agronomy, Department of Food Technology, Cara Dušana 34, 32000 Ţaţak, Serbia
Correspondence: *Department of Food Technology, Faculty of Agronomy, University of Kragujevac, Cara Dušana 34, 32000 Ţaţak, Republic of Serbia. Phone: +381 32 30 34 00; Fax: +381 32 30 34 01; Mobile: +381 64 358 85 47; e-mail address: [email protected] **Department of Biotechnology and Pharmaceutical Engineering, Faculty of Technology University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia Tel: +381 65 9577200; Fax: +381 21 450413; e-mail: [email protected]
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Abstract
Erica carnea L. or spring heath, is a perennial evergreen shrub, which belongs to the Ericaceae botanical family, which plant species are known for their biological activity and medical application. Despite the wide range of biological activity and medical application, Erica carnea L. has not been studied. This study deals with the application of conventional and non-conventional extraction approaches for isolation of bioactive compounds from the plant. Obtained extracts was tested regarding their chemical profile (total phenolics, flavonoids, condensed tannins, gallotannins and anthocyanins contents) and biological activity (antioxidant, cytotoxic and antibacterial activities). Phenolic profile of extracts was established using HPLC-DAD analysis where rosmarinic acid and rutin were dominant compounds. Results of antioxidant and cytotoxic activities demonstrated the domination of subcritical water extract, while ultrasound-assisted extract exhibited the highest total antibacterial activity. Presented results demonstrated that plant Erica carnea L. might be used as a potential source of biologically compounds.
Keywords: Erica carnea L., Extraction, Chemical composition, HPLC-DAD analysis, Biological activity
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1. Introduction
Erica carnea L. or spring heath, is a perennial evergreen shrub, which belongs to the Ericaceae botanical family and Erica genus [1]. This plant may grow up to 50 cm in height, leaves are in the form of thick, short and narrow needles, flowers are grouped into clusters of blossoms, usually longer on one side, while fruits are in the shape of capsule with the seeds inside it [2]. The plant itself is growing in Central and Southern Europe and South Africa in the mountains, in deciduous, coniferous or mixed forests, while in Serbia it grows in the Tara mountain [3]. Plant species of this family exert wide range of biological activities such as antioxidant [4,5], antidiabetic [6], anti-inflammatory [7], antibacterial [8] and analgesic [9]. Species of this botanical family have also found their application in medicine for the treatment of urinary tract infection [10]. Phenolic compounds, which are the product of secondary metabolism of plants, are one the most investigated class of natural products due to their wide range of biological activities, such as antioxidant, cytotoxic, antimicrobial, anti-inflammatory, antiulcer, antispasmodic, antiviral and many other activities [11–13]. There are over 8000 compounds which belong to one of the following group: simple phenolic, phenolic acids, stilbenes, flavonoids, coumarins, tannins and others [14,15]. For isolation of these and other compounds from their natural sources, many different approaches may be applied. Among them are conventional extraction techniques such as maceration and Soxhlet extraction and non-conventional techniques such as ultrasound-assisted, microwave-assisted and subcritical water extraction techniques [16– 18]. Every technique possesses certain advantages and disadvantages and they are usually combined to obtain improved results. Soxhlet extraction represents one of the conventional techniques which applies mainly toxic and environmental non-friendly solvents such as hexane and methylene chloride, but is also
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standard technique and main reference for evaluation of other extraction techniques performances [17]. To overcome usage of such approaches together with toxic solvents, and to increase total extraction yield and selectivity of the process as well, new and more environmental friendly extraction techniques such as ultrasound-assisted (UAE), microwaveassisted (MAE) techniques in combination with water and/or aqueous mixture with ethanol, as well as subcritical water (SCW) extraction technique have been developed [18]. Ultrasound extraction relies on application of ultrasound with the usual frequency from 20 to 100 MHz. This waves penetrate through a medium and create compression and expansion, thus producing the phenomenon called cavitation. On the other hand, microwave extraction relies on usage of microwaves, which frequency ranges from 300 to 300 GHz, thus creating uniform heating through the medium upon its direct impact on polar material. Process of heating is in correlation with the ionic conduction and dipole rotation mechanisms which occur during the interaction of the microwaves with the medium, thus creating the heat as a consequence of resistance of medium to flow ion [18–20]. As opposed to the previously described techniques, subcritical water extraction relays on application of water under the temperature above boiling point, while pressure maintains it in its liquid state. With the increasing in temperature, dielectric constant of water decreases, thus polarity of water consequently drops and may become similar to methanol [16,21,22]. Based on the previously conducted study, it has been expected that best results will be achieved using the SCW technique [23]. Despite the wide range of biological activity and medical application of plants from Ericaceae botanical family, Erica carnea L. is not in the focus of scientific community. There are only several studies which reported the useful properties of this plant [24,25]. Aim of this study was to apply conventional and non-conventional extraction techniques for isolation of biologically active compounds from this plant, to investigate biological activity of obtained
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extract and to establish their chemical profile. Obtained results were compared in order to evaluate efficiency of applied techniques and activities of obtained extracts.
2. Materials and methods
2.1 Chemicals and reagents
Folin-Ciocalteu reagent, aluminium chloride, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), rutin and potassium iodate (Sigma Chemical Company, St. Louis, SAD). All standard for HPLC analysis were of analytical grade and were purchased from Sigma chemicals Co (St Louis, MQ, USA) and Alfa aesar (Karlsruhe, Germany). Acetonitrile and phosphoric acid were of HPLC grade (Tedia Company, USA). Amracin (A) and cisdiamminedichloroplatinum (cis-DDP) were purchased from Tedia Company (USA). Ethanol and methanol were of analytical grade (Aldrich Chemical Co, Steinheim, Germany).
2.2 Plant material
Spring heath (Erica carnea L.) material was collected in Ţaţak area (Republic of Serbia) in 2011. Voucher specimens (Erica carnea L., Ţaţak area, determiner dr Milan Stankoviš, N° 126/017), are deposited at Institute of Biology, Faculty of Science, University of Kragujevac. Aerial parts of the plant were dried naturally in the shade on draft for one month. Dried plant material was grounded in the blender and kept in the paper bags before its usage.
2.3 Preparation of the extracts
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Soxhlet extraction was conducted in the following manner: plant material (75.0 g) was crushed and homogenized into small 3-5 mm pieces by a cylinder crusher and placed in the Soxhlet apparatus. Extraction process was carried out for eight hours using 96% ethanol as a solvent (600 mL) until the solvent discoloration (5 hours). Maceration was conducted using the following procedure: plant samples (10.0 g) were extracted using 96% ethanol (300 mL) as a solvent. The extraction process was carried out under the laboratory conditions at a temperature of 22 ºC in a sheltered, dry place for seven days, with occasional shaking to improve the maceration process. After seven days, extract was filtered through filter paper (Whatman, No.1) and concentrated to dry mass by a rotary evaporator (Devarot, Elektromedicina, Ljubljana, Slovenia) under vacuum and dried at 60ºC to the constant weight. The dried extracts were stored in a dark glass bottle at 4 ºC to prevent oxidative damage. Ultrasound-assisted extraction (UAE) was performed in ultrasonic water bath (EUP540A, Euinstruments, France). A sample (5g) was placed in volumetric flask and 100 mL of solvent (96% ethanol) was added. The mixture was sonificated for thirty minutes at frequency of 40 kHz and ultrasound power of 90% (216 W). Microwave-assisted extraction (MAE) was performed in domestic microwave oven, which was previously modified for this purpose. Extraction was conducted using the same sample weight, solvent volume and extraction time as in the case of ultrasound-assisted extraction. The extraction procedure program was as follows: one min pre-heating at 160 W; one min pre-heating at 320 W and thirty min extraction at 600 W. Subcritical water extraction (SWE) was performed in previously described home-made extractor system [26]. In all experimental runs, 5.0 g of plant sample was mixed with 100 mL of double-distilled water. Extraction was performed at pressure of 40 bars and temperature of 140 °C. Agitation was assured by vibrational movements of vessel platform at the frequency
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of 3Hz. Extraction duration in all experiments was 30 min. After the extraction, the process vessel was immediately cooled in flow-through water-bath at 20°C. Obtained extracts was filtered through filter paper (Whatman, No.1) and evaporated by a rotary evaporator (Devarot, Elektromedicina, Ljubljana, Slovenia) under vacuum and dried at 60 ºC to the constant weight. The dried extracts were stored in a dark glass bottle at 4 ºC to prevent oxidative damage. Yield of extraction techniques was determined by evaporating 10.00 mL of extract under vacuum and drying until the constant weight at 110 °C. Yield (Y) was expressed as grams of dry extract per 100 g of dry plant material (g/100 g DP).
2.4 Determination of total phenolics and flavonoids contents
Total phenolics (TPC) and total flavonoids (TFC) contents were determine using the previously described methods [27,28]. Final contents for TPC and TFC were expressed as milligrams of gallic acid equivalents per g of dry extract (mg GAE/g), while result for TFC were expressed as milligrams of rutin equivalents per g of dry extract (mg RU/g).
2.5 Determination of condensed tannins and gallotannins
Condensed tannins (CT) were determined according to previously described method which relies on the precipitation of proanthocyanidins with formaldehyde, while gallotannins (GA) were determined using the described potassium iodate assay [29]. Both contents were expressed as gallic acid equivalents.
2.6 Determination of anthocyanins
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Anthocyanins were determined according the previously described procedure [30,31] using pH single and differential methods. Total anthocyanins content (TAC) was expressed as cyanidin-3-glucoside per g of dry extract (mg C3G/g).
2.7 HPLC-DAD analysis
Quantification of individual phenolic compounds was performed using reversed phase HPLC analysis. The equipment used was an HPLC Agilent-1200 series with UV-Vis DAD detector for multi wavelength detection. After injecting 5 μL of sample, the separation was performed in an Agilent-Eclipse XDB C-18 column (4.6 x 150 mm), which was thermostated at 25ºC. Two solvents were used for the gradient elution: A (H2O+2%HCOOH) and B (80%ACN+2%HCOOH+H2O). The elution program used was as follows: from 0 to 10 min 0% B, from 10 to 28 min gradually increased 0-25% B, from 28 to 30 min 25% B, from 30 to 35 min gradually increased 25-50% B, from 35 to 40 min gradually increased 50-80% B, and finally for the last 5 min gradually decreased 80-0% B. Phenolic compounds in the samples were identified by comparing their retention times and spectra with retention time and spectra of standards for each component. Quantitative data were calculated from the calibration curves. Calibration curve, coefficient of correlation (R2), limit of detection (LOD) and limit of quantification (LOQ) are shown in Table 1. Content of phenolic compound were expressed as milligrams per gram of extract (mg/g).
2.8 Determination of biological activity
2.8.1 Determination of antioxidant activity
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Antioxidant activity of obtained extracts was determined using following, previously described assays: total antioxidant capacity [32], lipid peroxidation assay [33], hydroxyl radical scavenging activity [34] and DPPH radical scavenging activity [35] with slight modification [36]. Total antioxidant capacity (TA) was expressed as milligrams of ascorbic acid per gram of dry extract (mg AA/g). Results for lipid peroxidation assay were expressed as ILP50 in mg/mL, for hydroxyl radical scavenging activity as OH50 in mg/mL and for DPPH as IC50 in µg/mL. Results represent concentration of extract which inhibited 50% of peroxyl (ILP50), hydroxyl (OH50) and DPPH (IC50) radicals.
2.8.2 Antibacterial activity
The antibacterial activity of obtained extracts was tested in vitro against the following Grampositive bacteria: Staphylococcus saprophiticus ATCC 15035, Staphylococcus aureus ATOC 25923, Listeria ivanovii ATCC 19119, Listeria inocun ATCC 33090, Enterococcus faccalis ATCC 29212, Listeria monocytogenes ATCC 19112, Bacillus spieizeneii ATCC 6633 and Enterococcus faccium ATCC 6057, as well as against following Gram-negative bacteria: Escherichia coli ATCC 25922, Salmonella enteritidas ATCC 13076, Enterobacter aerogenus ATCC 13048, Citrobacter freundi ATCC 43864, Salmonella typhimurium ATCC 14028, Pseudomonas aeroginosa ATCC 27853 and Proteus mirabilis ATCC 35659. Antibacterial activity was estimated by measuring the minimum inhibitory concentrations (MIC). MICs of the extracts and cirsimarin against the test bacteria were determined by microdilution method in 96 multi-well microtiter plates according the previously described method [37]. The average of 3 values was calculated, and the obtained value was taken as the MIC for the tested sample and a standard drug (Amracin) [38].
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2.8.3 Cytotoxic activity
Cytotoxic activity was performed according the elsewhere described test [39], using earlier established MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay [40,41]. The following cell lines were used: RD (cell line derived from human rhabdomyosarcoma), Hep2c (cell line derived from human cervix carcinoma - HeLa derivative) and L2OB (cell line derived from murine fibroblast), against which activity of obtained extracts were measured. Results were expressed as IC50 values (µg/mL), which was defined as the concentration of an agent inhibiting cell survival by 50%, compared with a vehicle-treated control [42]. Standard compound was cis-diamminedichloroplatinum (cisDDP).
2.9 Statistical analysis
Statistical analysis was carried out using Statistica 6.0. (StatSoft Inc, Tulsa, OK, US). All experiments were performed at least in triplicate unless specified otherwise. Results are presented as a value ± standard deviation (SD). Levels of significance were as following: p < 0.1; p < 0.05 and p < 0.01.
3. Results and discussion
3.1 Chemical profile of obtained extracts
Results for Y, TPC, TFC, CT, GA and TAC obtained using spectrophotometric assays are presented in Table 2. According to the results, the highest contents of all compound classes
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was observed in SCW extract, while the lowest ones was in SE extract. Extraction yield (Y) was also the highest in SCW extract, which was actually expected regarding the previously conducted studies [23]. The amount of TPC obtained in SCW was about 50% higher than the corresponding value obtained by SE, while this percentage was slightly lower in the case of TFC, CT, GA and TAC. Results obtained for MAE and UAE extracts were slightly lower than those for SCW (about 10-20%), while MAC extract in some cases also achieved satisfactory results. Phenolic profile of obtained extracts was established applying the HPLC-DAD analysis, while the results are presented in Table 3, while chromatograms are presented in Figures 1s-5s (Supplementary data). Results showed that the highest content of quantified phenolic compounds was achieved in the SE extract, while the lowest content was observed in UAE extract. Dominant compound in SE was rosmarinic acid, which content was the highest regarding the all extracts. This compound represented 43.56% of all quantified compounds in SE extract, followed by quercetin (26.20%). Quercetin also consisted 24.28% of quantified compounds in MAC extract. Quercetin was the dominant in MAE extract and represented 33.79% of quantified compounds in this extract. On the other hand, rutin was the main quantified compound in all other extracts. This compound represented 27.66%, 58.95%, 30.69% and 30.07% of all quantified compounds in MAC, UAE, MAE and SCW extracts, respectively, but only 7.28% in SE extract. From the presented results, it might also be noticed that first five compounds were not determined in UAE extract. Luteolin-glycoside was not determined in SE, UAE and MAE, while apigenin-glycoside was determined in all extracts with the exception of MAC. Protocatechuic acid was note determined in any extract, while syringic acid was not presented in SE and MAC. Caffeic acid was observed in SE only, while chlorogenic acid was determined in SE and MAC.
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Reason for such diversity among the investigated extracts may be different mechanisms of thermal and mass transfers, as well as different solubility of compounds in the medium. Absence of phenolic acids in UAE may be explained by the degradation of compounds due to prolonged exposure to ultrasonic irradiation and/or heating effects [43]. On the other hand, absence of caffeic and chlorogenic acids in SCW extract was occurred due to low solubility of those compounds in the medium under the extraction conditions. It is well known that polarity of the water drops with the increasing in temperature. Thus, under the investigation conditions, less polar compounds should be better extracted, which is actual case [22]. Degradation of the compounds due to prolonged exposure to the irradiation and/or thermal processes might be the reason of absence of phenolic acids in the case of MAE extract. There are also some disagreements in the results obtained for TPC and HPLC-DAD. Although TPC was the highest in SCW extract (Table 2), HPLC-DAD analysis revealed that the highest content of phenolic compounds was in SE (Table 3). Such results may be the consequence of poor selectivity of Folin-Ciocalteu (F-C) reagent, which is even not selective toward antioxidant compounds [44,45]. It was indicated that other classes of compounds such as carbohydrates, amino acids, nucleotides, thiols, unsaturated fatty acids, proteins, vitamins, amines, aldehydes and ketones may be the possible contributors to overall results of this spectrophotometric test [46,47]. Beside the low selectivity of F-C reagent, occurrence of reaction under the extraction conditions (elevated temperature) such as Maillard reaction and caramelization, may produce new compounds which could react with the F-C reagent increasing the overall results of spectrophotometric tests, especially in the case of SCW and MAE [48–50].
3.2 Biological activity of obtained extract
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Biological activity of obtained extract was investigated regarding the antioxidant, cytotoxic and antibacterial activities. Antioxidant activity was established using four different assays: total antioxidant capacity, inhibition of lipid peroxidation, hydroxy radical scavenging and DPPH scavenging activities, while the results are presented in Table 4. The highest activity in all four tested was observed for the SCW extract, while the lowest was detected in the case of SE extract. MAE and UAE extracts showed similar activities in lipid peroxidation test, hydroxy radical scavenging and DPPH scavenging activities, while difference between activities was slightly higher in the case of total antioxidant capacity assay. On the other hand, similar activity between MAC and SE was observed in the case of total antioxidant capacity, lipid peroxidation test and hydroxy radical scavenging, while difference was slightly higher in the case of DPPH assay. Domination of SCW and MAE extracts regarding the antioxidant activity might be explained with the above-mentioned production of new compounds as a consequence of Maillard reaction and caramelization. These compounds might act as neo/antioxidants thus providing the highest antioxidant capacity to the processes at elevated temperature such as SCW and MAE [48–50]. Cytotoxic activity of obtained extract is presented in Table 5. Three different cell lines were used for assessment, and SCW extract again exhibited the strongest activity. MAE and UAE extracts showed similar activities, while tested cell lines were the least susceptible to the SE extract. Analysis of cell line sensitivity on tested extracts revealed that L2OB cells were the most sensitive to MAC and UAE extracts. SE and MAE extracts exhibited the highest potency against RD cells, while Hep2c cells were the most sensitive to SCW extract. Pearson’s correlation coefficients among TPC, TFC, CT, GA, TAC, TA, ILP50, OH50, IC50, Hep2c, RD and L2OB are presented in Table 6. Correlation coefficients are characterized as
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particularly high (r > 0.9), high (0.9 > r > 0.7), moderate (0.7 > r > 0.5) and poor correlation (r < 0.5). Correlation analysis showed that correlation among the most tested parameters were particularly high (r > 0.9). Correlations among TPC and ILP50, Hep2c and TFC, GA, TA, OH50, IC50, and RD, as well as among L2OB and ILP50 were high with r > 0.8 which represents quite high correlation. Correlations between TAC and ILP50, Hep2c and TPC, CT and L2OB, as well as among ILP50 and TPC were high with the 0.8 > r > 0.7 which may be accepted as good correlation, while the correlation between Hep2c and TAC was moderate (r = 0.6792). Results revealed high correlation between the contents of the compounds and biological activities of obtained extracts. Correlation among performed assays, as well as contents of compounds were also high. This may indicate the close connection between phenolic compounds and exerted biological activity of prepared extracts. Antibacterial activity of prepared extracts was established against fifteen different bacterial strains, while the results are presented in Table 7. Generally, the best result was observed in the case of UAE extract, while MAE and SCW extract exhibited similar activities. The strongest activity was exerted by SE against E. coli, MAC against E. aerogenus and P. mirabilis, UAE against S. typhimurium and SCW against S. saprophiticus with the MIC value of 7.81 µg/mL. It is known that plant synthetized phenolic compound in response to microbial infection , thus their presence in extracts might be the explanation for antibacterial activity [13,51,52]. Mori et al. [51] indicated that free hydroxyl group attached to the phenyl ring of flavonoids are necessary for activity. But there are some indications that phenolic compounds are not the only responsible for antibacterial activity. Other secondary metabolites as well as synergistic effect might be involved here and responsible for such behavior [13]. This may be the explanation for high activity of UAE extract against most of the tested strains. Further
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research is needed in order to establish the relationship between chemical composition of extracts and their antibacterial activity.
4. Conclusion
The preset study showed that Erica carnea L. possesses a good potential to be used as a source of biologically active compounds. Based on the presented results, it may be noticed that prepared extracts exhibited high antioxidant, cytotoxic and antibacterial activity. Subcritical water extract generally showed the best properties, but other non-conventional techniques demonstrated satisfactory results. Using modern analytical technique, such as HPLC-DAD, presence of the certain phenolic compounds has been confirmed. Analysis showed that amount of the compounds in the extract depends on the applied extraction technique as well as on the experimental conditions. All in all, obtained results in this study should encourage further and deeper investigation of this plant together with the new fields and possibilities for its application.
Acknowledgements
Authors of this study are grateful to Dr Milan Stankoviš, University of Kragujevac, Faculty of Sciences, Institute of Biology, Republic of Serbia, for support in terms of confirmation and deposition of Erica carnea L.
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Table 1. Analytical parameters for 18 phenolic compounds used for HPLC-DAD analysis Calibration (R2) RT* LOD** curve (min) μg/ml Protocatechuic acid y=13307.5x+0.235 0.9988 12.52 0.004 p-Hydroxybenzoic acid y=9934.4x+0.086 0.9998 17.65 0.003 Caffeic acid Y=32241.5-0.154 1.0000 21.19 0.009 Vanillic acid Y=10781.0+0.520 0.9995 22.15 0.003 Chlorogenic acid Y=10491.8-0.525 0.9998 22.25 0.035 Syringic acid Y=11253.6+0.465 0.9999 23.88 0.011 p-Coumaric acid Y=16239.7-0.832 0.9997 24.83 0.042 Ferulic acid Y=24685.7-0.754 0.9998 27.35 0.029 Sinapic acid Y=13332.5+1.062 0.9998 29.11 0.032 Rutin Y=4589.0-0.674 0.9999 29.30 0.052 Luteolin-glycoside Y=13132.5+1.042 0.9998 31.22 0.032 Apigenin-glycoside Y=4289.0-0.674 0.9999 32.21 0.052 Rosmarinic acid Y=5385.2+0.656 0.9998 33.58 0.018 Quercetin Y=10336.2+0.320 0.9996 36.50 0.033 Luteolin Y=15958.6-0.234 0.9998 37.31 0.030 Naringenin Y=14797.5+0.980 1.0000 37.85 0.055 Kaempferol Y=13636.5+0.098 0.9997 38.29 0.029 Apigenin Y=6229.8+1.200 0.9996 40.10 0.045 *RT-retention time, **LOD-limit of detection, ***LOQ-limit of quantification. Compound
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LOQ*** μg/ml 0.013 0.010 0.030 0.010 0.116 0.037 0.140 0.097 0.106 0.173 0.106 0.173 0.060 0.110 0.100 0.183 0.097 0.150
Table 2. Chemical profile of Erica carnea L. extracts obtained by spectrophotometric assays Extract SE
Y (g/100 g DP) 12.10
MAC
15.30
UAE
18.33
MAE
30.65
SCW
42.66
TPC (mg GAE/g) 98.86 ± 0.49 118.14 ± 0.26 127.42 ± 0.87 135.56 ± 0.19 144.12 ± 0.23
TFC (mg RU/g) 19.18 ± 0.37 20.57 ± 0.58 22.55 ± 0.17 23.81 ± 0.60 26.24 ± 0.18
23
CT (mg GAE/g) 55.85 ± 0.44 57.19 ± 0.37 60.65 ± 0.54 61.70 ± 0.19 62.53 ± 0.81
GA (mg GAE/g) 17.13 ± 0.94 19.74 ± 0.96 20.19 ± 0.42 23.36 ± 0.77 25.56 ± 0.64
TAC (mg C3G/g) 10.60 ± 0.40 11.86 ± 0.73 12.46 ± 0.19 13.07 ± 0.54 13.13 ± 0.28
Table 3. Polyphenolic profile of Erica carnea L. extracts Compound Protocatechuic acid p-Hydroxybenzoic acid Caffeic acid Vanillic acid Chlorogenic acid Syringic acid p-Coumaric acid Ferulic acid Sinapic acid Rutin Luteolin-glycoside Apigenin-glycoside Rosmarinic acid Quercetin Luteolin Naringenin Kaempferol Apigenin Σ *ND-not determined.
SE ND* 0.102 0.287 0.293 1.217 ND 0.553 0.393 0.238 1.987 ND 0.362 11.890 7.151 0.181 0.306 0.985 1.351 27.296
Content (mg/g) MAC UAE ND ND 0.770 ND ND ND 0.129 ND 0.132 ND ND 0.017 0.108 0.015 0.113 0.022 0.408 0.123 2.159 0.728 0.336 ND ND 0.016 0.746 0.038 1.895 0.149 0.559 0.058 0.055 0.019 0.074 0.032 0.321 0.018 7.805 1.235
24
MAE ND 0.121 ND ND ND 0.053 0.044 0.042 0.267 0.908 ND 0.088 0.131 1.000 0.100 0.044 0.105 0.179 2.959
SCW ND 0.148 ND 0.086 ND 0.048 0.054 0.032 0.099 0.397 0.033 0.024 0.036 0.282 0.016 0.013 0.023 0.029 1.320
Table 4. Antioxidant activity of obtained Erica carnea L. extracts Extract SE MAC UAE MAE SCW
TA (µg AA/G) 112.34 ± 0.43 119.92 ± 0.48 134.71 ± 0.29 159.09 ± 0.82 171.32 ± 0.87
ILP50 (µg/mL) 31.35 ± 0.42 30.73 ± 0.08 28.57 ± 0.44 27.56 ± 0.81 21.71 ± 0.45
25
OH50 (µg/mL) 33.51 ± 0.88 30.15 ± 0.93 26.83 ± 0.77 24.41 ± 0.81 20.63 ± 0.94
IC50 (µg/mL) 46.68 ± 1.01 37.83 ± 0.98 36.47 ± 0.69 30,60 ± 0.99 23.73 ± 0.53
Table 5. Cytotoxic activity of obtained Erica carnea L. extracts Extract
IC50 (µg/mL) RD cells 32.43 ± 0.46 30.84 ± 0.28 23.54 ± 0.48 19.23 ± 0.79 16.17 ± 0.35 1.4 ± 0.97
Hep2c cells SE 34.26 ± 0.37 MAC 32.28 ± 0.44 UAE 31.70 ± 0.89 MAE 27.54 ± 0.32 SCW 11.54 ± 0.55 cis-DDP* 0.94 ± 0.55 *cis-DDP, cis-diamminedichloroplatinum.
26
L2OB cells 33.09 ± 0.65 28.51 ± 0.14 22.63 ± 0.49 20.09 ± 0.60 18.51 ± 0.52 0.72 ± 0.64
Table 6. Pearson’s correlation coefficients among TPC, TFC, CT, GA, TAC, TA, ILP50, OH50, IC50, Hep2c, RD and L2OB Test TPC TFC CT GA TAC TA ILP50 OH50 IC50 Hep2c RD L2OB TPC 1 TFC 0.9650a 1 CT 0.9620a 0.9596a 1 a a GA 0.9594 0.9743 0.9125b 1 TAC 0.9867a 0.9200b 0.9568b 0.9217b 1 b a b a TA 0.9387 0.9794 0.9477 0.9789 0.9073b 1 ILP50 -0.8609c -0.9567b -0.8483c -0.9223b -0.7727d -0.9213b 1 a a a a b a OH50 -0.9819 -0.9971 -0.9677 -0.9781 -0.9463 -0.9765 0.9352b 1 IC50 -0.9781a -0.9749a -0.9156b -0.9916a -0.9395b -0.9558b 0.9186b 0.9829a 1 d b d b d c a c Hep2c -0.7839 -0.8942 -0.7355 -0.8899 -0.6782 -0.8644 0.9783 0.8681 0.8808b 1 RD -0.9476b -0.9798a -0.9860a -0.9448b -0.9256b -0.9838a 0.9018b 0.9787a 0.9320b 0.8119c 1 L2OB -0.9833a -0.9590a -0.9941a -0.9284b -0.9820a -0.9435b 0.8375a 0.9733a 0.9383b 0.7321d 0.9743a 1 a b c d statistically significant at p < 0.01; statistically significant at p < 0.05; statistically significant at p < 0.1, statistically insignificant
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Table 7. Antibacterial activity of obtained Erica carnea L. extracts Strain S. saprophiticus ATCC 15035 S. aureus ATCC 25923 L. ivanovii ATCC 19119 L. inocun ATCC 33090 E. faccalis ATCC 2912 L. monocytogenes ATCC 19112 B. spieizeneii ATCC 6633 E. faccium ATCC 6057 E. coli ATCC 25922 S. enteritidas ATCC 13076 E. aerogenus ATCC 13048 C. freundi ATCC 43864 S. typhimurium ATCC 14028 P. aeroginosa ATCC 27853 P. mirabilis ATCC 35659 *A-Amracin.
SE
MAC
MIC (µg/mL) UAE MAE
31.25
15.82
15.82
31.25
7.81
0.24
250.00
250.00
31.25
62.50
62.50
0.97
31.25
500.00
31.25
250.00
250.00
0.49
125.00
125.00
15.82
125.00
125.00
0.97
62.50
250.00
15.82
250.00
250.00
0.49
125.00
500.00
31.25
62.50
62.50
0.49
125.00
125.00
31.25
250.00
250.00
0.24
250.00
62.50
31.25
500.00
125.00
0.97
7.81
62.50
62.50
125.00
250.00
0.49
125.00
250.00
250.00
250.00
62.50
0.97
62.50
7.81
15.82
500.00
250.00
0.49
125.00
62.50
15.82
250.00
125.00
0.49
31.25
15.82
7.81
500.00
250.00
0.24
125.00
125.00
15.82
62.50
62.50
0.97
250.00
7.81
62.50
250.00
250.00
0.49
28
SCW
A*
Graphical Abstract
29
S0896-8446(17)30033-5 http://dx.doi.org/doi:10.1016/j.supflu.2017.03.023 SUPFLU 3892
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
12-1-2017 25-3-2017 27-3-2017
Please cite this article as: Vesna Veliˇckovi´c, Saˇsa Ðurovi´c, Marija Radojkovi´c, ˇ Aleksandra Cvetanovi´c, Jaroslava Svarc-Gaji´ c, Jelena Vuji´c, Sre´cko Trifunovi´c, Pavle Z.Maˇskovi´c, Application of conventional and non-conventional extraction approaches for extraction of Erica carnea L.: chemical profile and biological activity of obtained extracts, The Journal of Supercritical Fluidshttp://dx.doi.org/10.1016/j.supflu.2017.03.023 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.
Application of conventional and non-conventional extraction approaches for extraction of Erica carnea L.: chemical profile and biological activity of obtained extracts
Vesna Veliţkoviš1, Saša Đuroviš2**, Marija Radojkoviš2, Aleksandra Cvetanoviš2, Jaroslava Švarc-Gajiš2, Jelena Vujiš1, Sreško Trifunoviš1, Pavle Z. Maškoviš3*
1
University of Kragujevac, Faculty of Science, Department of Chemistry, Radoja Domanoviša 12, 34000 Kragujevac, Serbia
2
University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
3
University of Kragujevac, Faculty of Agronomy, Department of Food Technology, Cara Dušana 34, 32000 Ţaţak, Serbia
Correspondence: *Department of Food Technology, Faculty of Agronomy, University of Kragujevac, Cara Dušana 34, 32000 Ţaţak, Republic of Serbia. Phone: +381 32 30 34 00; Fax: +381 32 30 34 01; Mobile: +381 64 358 85 47; e-mail address: [email protected] **Department of Biotechnology and Pharmaceutical Engineering, Faculty of Technology University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia Tel: +381 65 9577200; Fax: +381 21 450413; e-mail: [email protected]
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Abstract
Erica carnea L. or spring heath, is a perennial evergreen shrub, which belongs to the Ericaceae botanical family, which plant species are known for their biological activity and medical application. Despite the wide range of biological activity and medical application, Erica carnea L. has not been studied. This study deals with the application of conventional and non-conventional extraction approaches for isolation of bioactive compounds from the plant. Obtained extracts was tested regarding their chemical profile (total phenolics, flavonoids, condensed tannins, gallotannins and anthocyanins contents) and biological activity (antioxidant, cytotoxic and antibacterial activities). Phenolic profile of extracts was established using HPLC-DAD analysis where rosmarinic acid and rutin were dominant compounds. Results of antioxidant and cytotoxic activities demonstrated the domination of subcritical water extract, while ultrasound-assisted extract exhibited the highest total antibacterial activity. Presented results demonstrated that plant Erica carnea L. might be used as a potential source of biologically compounds.
Keywords: Erica carnea L., Extraction, Chemical composition, HPLC-DAD analysis, Biological activity
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1. Introduction
Erica carnea L. or spring heath, is a perennial evergreen shrub, which belongs to the Ericaceae botanical family and Erica genus [1]. This plant may grow up to 50 cm in height, leaves are in the form of thick, short and narrow needles, flowers are grouped into clusters of blossoms, usually longer on one side, while fruits are in the shape of capsule with the seeds inside it [2]. The plant itself is growing in Central and Southern Europe and South Africa in the mountains, in deciduous, coniferous or mixed forests, while in Serbia it grows in the Tara mountain [3]. Plant species of this family exert wide range of biological activities such as antioxidant [4,5], antidiabetic [6], anti-inflammatory [7], antibacterial [8] and analgesic [9]. Species of this botanical family have also found their application in medicine for the treatment of urinary tract infection [10]. Phenolic compounds, which are the product of secondary metabolism of plants, are one the most investigated class of natural products due to their wide range of biological activities, such as antioxidant, cytotoxic, antimicrobial, anti-inflammatory, antiulcer, antispasmodic, antiviral and many other activities [11–13]. There are over 8000 compounds which belong to one of the following group: simple phenolic, phenolic acids, stilbenes, flavonoids, coumarins, tannins and others [14,15]. For isolation of these and other compounds from their natural sources, many different approaches may be applied. Among them are conventional extraction techniques such as maceration and Soxhlet extraction and non-conventional techniques such as ultrasound-assisted, microwave-assisted and subcritical water extraction techniques [16– 18]. Every technique possesses certain advantages and disadvantages and they are usually combined to obtain improved results. Soxhlet extraction represents one of the conventional techniques which applies mainly toxic and environmental non-friendly solvents such as hexane and methylene chloride, but is also
3
standard technique and main reference for evaluation of other extraction techniques performances [17]. To overcome usage of such approaches together with toxic solvents, and to increase total extraction yield and selectivity of the process as well, new and more environmental friendly extraction techniques such as ultrasound-assisted (UAE), microwaveassisted (MAE) techniques in combination with water and/or aqueous mixture with ethanol, as well as subcritical water (SCW) extraction technique have been developed [18]. Ultrasound extraction relies on application of ultrasound with the usual frequency from 20 to 100 MHz. This waves penetrate through a medium and create compression and expansion, thus producing the phenomenon called cavitation. On the other hand, microwave extraction relies on usage of microwaves, which frequency ranges from 300 to 300 GHz, thus creating uniform heating through the medium upon its direct impact on polar material. Process of heating is in correlation with the ionic conduction and dipole rotation mechanisms which occur during the interaction of the microwaves with the medium, thus creating the heat as a consequence of resistance of medium to flow ion [18–20]. As opposed to the previously described techniques, subcritical water extraction relays on application of water under the temperature above boiling point, while pressure maintains it in its liquid state. With the increasing in temperature, dielectric constant of water decreases, thus polarity of water consequently drops and may become similar to methanol [16,21,22]. Based on the previously conducted study, it has been expected that best results will be achieved using the SCW technique [23]. Despite the wide range of biological activity and medical application of plants from Ericaceae botanical family, Erica carnea L. is not in the focus of scientific community. There are only several studies which reported the useful properties of this plant [24,25]. Aim of this study was to apply conventional and non-conventional extraction techniques for isolation of biologically active compounds from this plant, to investigate biological activity of obtained
4
extract and to establish their chemical profile. Obtained results were compared in order to evaluate efficiency of applied techniques and activities of obtained extracts.
2. Materials and methods
2.1 Chemicals and reagents
Folin-Ciocalteu reagent, aluminium chloride, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), rutin and potassium iodate (Sigma Chemical Company, St. Louis, SAD). All standard for HPLC analysis were of analytical grade and were purchased from Sigma chemicals Co (St Louis, MQ, USA) and Alfa aesar (Karlsruhe, Germany). Acetonitrile and phosphoric acid were of HPLC grade (Tedia Company, USA). Amracin (A) and cisdiamminedichloroplatinum (cis-DDP) were purchased from Tedia Company (USA). Ethanol and methanol were of analytical grade (Aldrich Chemical Co, Steinheim, Germany).
2.2 Plant material
Spring heath (Erica carnea L.) material was collected in Ţaţak area (Republic of Serbia) in 2011. Voucher specimens (Erica carnea L., Ţaţak area, determiner dr Milan Stankoviš, N° 126/017), are deposited at Institute of Biology, Faculty of Science, University of Kragujevac. Aerial parts of the plant were dried naturally in the shade on draft for one month. Dried plant material was grounded in the blender and kept in the paper bags before its usage.
2.3 Preparation of the extracts
5
Soxhlet extraction was conducted in the following manner: plant material (75.0 g) was crushed and homogenized into small 3-5 mm pieces by a cylinder crusher and placed in the Soxhlet apparatus. Extraction process was carried out for eight hours using 96% ethanol as a solvent (600 mL) until the solvent discoloration (5 hours). Maceration was conducted using the following procedure: plant samples (10.0 g) were extracted using 96% ethanol (300 mL) as a solvent. The extraction process was carried out under the laboratory conditions at a temperature of 22 ºC in a sheltered, dry place for seven days, with occasional shaking to improve the maceration process. After seven days, extract was filtered through filter paper (Whatman, No.1) and concentrated to dry mass by a rotary evaporator (Devarot, Elektromedicina, Ljubljana, Slovenia) under vacuum and dried at 60ºC to the constant weight. The dried extracts were stored in a dark glass bottle at 4 ºC to prevent oxidative damage. Ultrasound-assisted extraction (UAE) was performed in ultrasonic water bath (EUP540A, Euinstruments, France). A sample (5g) was placed in volumetric flask and 100 mL of solvent (96% ethanol) was added. The mixture was sonificated for thirty minutes at frequency of 40 kHz and ultrasound power of 90% (216 W). Microwave-assisted extraction (MAE) was performed in domestic microwave oven, which was previously modified for this purpose. Extraction was conducted using the same sample weight, solvent volume and extraction time as in the case of ultrasound-assisted extraction. The extraction procedure program was as follows: one min pre-heating at 160 W; one min pre-heating at 320 W and thirty min extraction at 600 W. Subcritical water extraction (SWE) was performed in previously described home-made extractor system [26]. In all experimental runs, 5.0 g of plant sample was mixed with 100 mL of double-distilled water. Extraction was performed at pressure of 40 bars and temperature of 140 °C. Agitation was assured by vibrational movements of vessel platform at the frequency
6
of 3Hz. Extraction duration in all experiments was 30 min. After the extraction, the process vessel was immediately cooled in flow-through water-bath at 20°C. Obtained extracts was filtered through filter paper (Whatman, No.1) and evaporated by a rotary evaporator (Devarot, Elektromedicina, Ljubljana, Slovenia) under vacuum and dried at 60 ºC to the constant weight. The dried extracts were stored in a dark glass bottle at 4 ºC to prevent oxidative damage. Yield of extraction techniques was determined by evaporating 10.00 mL of extract under vacuum and drying until the constant weight at 110 °C. Yield (Y) was expressed as grams of dry extract per 100 g of dry plant material (g/100 g DP).
2.4 Determination of total phenolics and flavonoids contents
Total phenolics (TPC) and total flavonoids (TFC) contents were determine using the previously described methods [27,28]. Final contents for TPC and TFC were expressed as milligrams of gallic acid equivalents per g of dry extract (mg GAE/g), while result for TFC were expressed as milligrams of rutin equivalents per g of dry extract (mg RU/g).
2.5 Determination of condensed tannins and gallotannins
Condensed tannins (CT) were determined according to previously described method which relies on the precipitation of proanthocyanidins with formaldehyde, while gallotannins (GA) were determined using the described potassium iodate assay [29]. Both contents were expressed as gallic acid equivalents.
2.6 Determination of anthocyanins
7
Anthocyanins were determined according the previously described procedure [30,31] using pH single and differential methods. Total anthocyanins content (TAC) was expressed as cyanidin-3-glucoside per g of dry extract (mg C3G/g).
2.7 HPLC-DAD analysis
Quantification of individual phenolic compounds was performed using reversed phase HPLC analysis. The equipment used was an HPLC Agilent-1200 series with UV-Vis DAD detector for multi wavelength detection. After injecting 5 μL of sample, the separation was performed in an Agilent-Eclipse XDB C-18 column (4.6 x 150 mm), which was thermostated at 25ºC. Two solvents were used for the gradient elution: A (H2O+2%HCOOH) and B (80%ACN+2%HCOOH+H2O). The elution program used was as follows: from 0 to 10 min 0% B, from 10 to 28 min gradually increased 0-25% B, from 28 to 30 min 25% B, from 30 to 35 min gradually increased 25-50% B, from 35 to 40 min gradually increased 50-80% B, and finally for the last 5 min gradually decreased 80-0% B. Phenolic compounds in the samples were identified by comparing their retention times and spectra with retention time and spectra of standards for each component. Quantitative data were calculated from the calibration curves. Calibration curve, coefficient of correlation (R2), limit of detection (LOD) and limit of quantification (LOQ) are shown in Table 1. Content of phenolic compound were expressed as milligrams per gram of extract (mg/g).
2.8 Determination of biological activity
2.8.1 Determination of antioxidant activity
8
Antioxidant activity of obtained extracts was determined using following, previously described assays: total antioxidant capacity [32], lipid peroxidation assay [33], hydroxyl radical scavenging activity [34] and DPPH radical scavenging activity [35] with slight modification [36]. Total antioxidant capacity (TA) was expressed as milligrams of ascorbic acid per gram of dry extract (mg AA/g). Results for lipid peroxidation assay were expressed as ILP50 in mg/mL, for hydroxyl radical scavenging activity as OH50 in mg/mL and for DPPH as IC50 in µg/mL. Results represent concentration of extract which inhibited 50% of peroxyl (ILP50), hydroxyl (OH50) and DPPH (IC50) radicals.
2.8.2 Antibacterial activity
The antibacterial activity of obtained extracts was tested in vitro against the following Grampositive bacteria: Staphylococcus saprophiticus ATCC 15035, Staphylococcus aureus ATOC 25923, Listeria ivanovii ATCC 19119, Listeria inocun ATCC 33090, Enterococcus faccalis ATCC 29212, Listeria monocytogenes ATCC 19112, Bacillus spieizeneii ATCC 6633 and Enterococcus faccium ATCC 6057, as well as against following Gram-negative bacteria: Escherichia coli ATCC 25922, Salmonella enteritidas ATCC 13076, Enterobacter aerogenus ATCC 13048, Citrobacter freundi ATCC 43864, Salmonella typhimurium ATCC 14028, Pseudomonas aeroginosa ATCC 27853 and Proteus mirabilis ATCC 35659. Antibacterial activity was estimated by measuring the minimum inhibitory concentrations (MIC). MICs of the extracts and cirsimarin against the test bacteria were determined by microdilution method in 96 multi-well microtiter plates according the previously described method [37]. The average of 3 values was calculated, and the obtained value was taken as the MIC for the tested sample and a standard drug (Amracin) [38].
9
2.8.3 Cytotoxic activity
Cytotoxic activity was performed according the elsewhere described test [39], using earlier established MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay [40,41]. The following cell lines were used: RD (cell line derived from human rhabdomyosarcoma), Hep2c (cell line derived from human cervix carcinoma - HeLa derivative) and L2OB (cell line derived from murine fibroblast), against which activity of obtained extracts were measured. Results were expressed as IC50 values (µg/mL), which was defined as the concentration of an agent inhibiting cell survival by 50%, compared with a vehicle-treated control [42]. Standard compound was cis-diamminedichloroplatinum (cisDDP).
2.9 Statistical analysis
Statistical analysis was carried out using Statistica 6.0. (StatSoft Inc, Tulsa, OK, US). All experiments were performed at least in triplicate unless specified otherwise. Results are presented as a value ± standard deviation (SD). Levels of significance were as following: p < 0.1; p < 0.05 and p < 0.01.
3. Results and discussion
3.1 Chemical profile of obtained extracts
Results for Y, TPC, TFC, CT, GA and TAC obtained using spectrophotometric assays are presented in Table 2. According to the results, the highest contents of all compound classes
10
was observed in SCW extract, while the lowest ones was in SE extract. Extraction yield (Y) was also the highest in SCW extract, which was actually expected regarding the previously conducted studies [23]. The amount of TPC obtained in SCW was about 50% higher than the corresponding value obtained by SE, while this percentage was slightly lower in the case of TFC, CT, GA and TAC. Results obtained for MAE and UAE extracts were slightly lower than those for SCW (about 10-20%), while MAC extract in some cases also achieved satisfactory results. Phenolic profile of obtained extracts was established applying the HPLC-DAD analysis, while the results are presented in Table 3, while chromatograms are presented in Figures 1s-5s (Supplementary data). Results showed that the highest content of quantified phenolic compounds was achieved in the SE extract, while the lowest content was observed in UAE extract. Dominant compound in SE was rosmarinic acid, which content was the highest regarding the all extracts. This compound represented 43.56% of all quantified compounds in SE extract, followed by quercetin (26.20%). Quercetin also consisted 24.28% of quantified compounds in MAC extract. Quercetin was the dominant in MAE extract and represented 33.79% of quantified compounds in this extract. On the other hand, rutin was the main quantified compound in all other extracts. This compound represented 27.66%, 58.95%, 30.69% and 30.07% of all quantified compounds in MAC, UAE, MAE and SCW extracts, respectively, but only 7.28% in SE extract. From the presented results, it might also be noticed that first five compounds were not determined in UAE extract. Luteolin-glycoside was not determined in SE, UAE and MAE, while apigenin-glycoside was determined in all extracts with the exception of MAC. Protocatechuic acid was note determined in any extract, while syringic acid was not presented in SE and MAC. Caffeic acid was observed in SE only, while chlorogenic acid was determined in SE and MAC.
11
Reason for such diversity among the investigated extracts may be different mechanisms of thermal and mass transfers, as well as different solubility of compounds in the medium. Absence of phenolic acids in UAE may be explained by the degradation of compounds due to prolonged exposure to ultrasonic irradiation and/or heating effects [43]. On the other hand, absence of caffeic and chlorogenic acids in SCW extract was occurred due to low solubility of those compounds in the medium under the extraction conditions. It is well known that polarity of the water drops with the increasing in temperature. Thus, under the investigation conditions, less polar compounds should be better extracted, which is actual case [22]. Degradation of the compounds due to prolonged exposure to the irradiation and/or thermal processes might be the reason of absence of phenolic acids in the case of MAE extract. There are also some disagreements in the results obtained for TPC and HPLC-DAD. Although TPC was the highest in SCW extract (Table 2), HPLC-DAD analysis revealed that the highest content of phenolic compounds was in SE (Table 3). Such results may be the consequence of poor selectivity of Folin-Ciocalteu (F-C) reagent, which is even not selective toward antioxidant compounds [44,45]. It was indicated that other classes of compounds such as carbohydrates, amino acids, nucleotides, thiols, unsaturated fatty acids, proteins, vitamins, amines, aldehydes and ketones may be the possible contributors to overall results of this spectrophotometric test [46,47]. Beside the low selectivity of F-C reagent, occurrence of reaction under the extraction conditions (elevated temperature) such as Maillard reaction and caramelization, may produce new compounds which could react with the F-C reagent increasing the overall results of spectrophotometric tests, especially in the case of SCW and MAE [48–50].
3.2 Biological activity of obtained extract
12
Biological activity of obtained extract was investigated regarding the antioxidant, cytotoxic and antibacterial activities. Antioxidant activity was established using four different assays: total antioxidant capacity, inhibition of lipid peroxidation, hydroxy radical scavenging and DPPH scavenging activities, while the results are presented in Table 4. The highest activity in all four tested was observed for the SCW extract, while the lowest was detected in the case of SE extract. MAE and UAE extracts showed similar activities in lipid peroxidation test, hydroxy radical scavenging and DPPH scavenging activities, while difference between activities was slightly higher in the case of total antioxidant capacity assay. On the other hand, similar activity between MAC and SE was observed in the case of total antioxidant capacity, lipid peroxidation test and hydroxy radical scavenging, while difference was slightly higher in the case of DPPH assay. Domination of SCW and MAE extracts regarding the antioxidant activity might be explained with the above-mentioned production of new compounds as a consequence of Maillard reaction and caramelization. These compounds might act as neo/antioxidants thus providing the highest antioxidant capacity to the processes at elevated temperature such as SCW and MAE [48–50]. Cytotoxic activity of obtained extract is presented in Table 5. Three different cell lines were used for assessment, and SCW extract again exhibited the strongest activity. MAE and UAE extracts showed similar activities, while tested cell lines were the least susceptible to the SE extract. Analysis of cell line sensitivity on tested extracts revealed that L2OB cells were the most sensitive to MAC and UAE extracts. SE and MAE extracts exhibited the highest potency against RD cells, while Hep2c cells were the most sensitive to SCW extract. Pearson’s correlation coefficients among TPC, TFC, CT, GA, TAC, TA, ILP50, OH50, IC50, Hep2c, RD and L2OB are presented in Table 6. Correlation coefficients are characterized as
13
particularly high (r > 0.9), high (0.9 > r > 0.7), moderate (0.7 > r > 0.5) and poor correlation (r < 0.5). Correlation analysis showed that correlation among the most tested parameters were particularly high (r > 0.9). Correlations among TPC and ILP50, Hep2c and TFC, GA, TA, OH50, IC50, and RD, as well as among L2OB and ILP50 were high with r > 0.8 which represents quite high correlation. Correlations between TAC and ILP50, Hep2c and TPC, CT and L2OB, as well as among ILP50 and TPC were high with the 0.8 > r > 0.7 which may be accepted as good correlation, while the correlation between Hep2c and TAC was moderate (r = 0.6792). Results revealed high correlation between the contents of the compounds and biological activities of obtained extracts. Correlation among performed assays, as well as contents of compounds were also high. This may indicate the close connection between phenolic compounds and exerted biological activity of prepared extracts. Antibacterial activity of prepared extracts was established against fifteen different bacterial strains, while the results are presented in Table 7. Generally, the best result was observed in the case of UAE extract, while MAE and SCW extract exhibited similar activities. The strongest activity was exerted by SE against E. coli, MAC against E. aerogenus and P. mirabilis, UAE against S. typhimurium and SCW against S. saprophiticus with the MIC value of 7.81 µg/mL. It is known that plant synthetized phenolic compound in response to microbial infection , thus their presence in extracts might be the explanation for antibacterial activity [13,51,52]. Mori et al. [51] indicated that free hydroxyl group attached to the phenyl ring of flavonoids are necessary for activity. But there are some indications that phenolic compounds are not the only responsible for antibacterial activity. Other secondary metabolites as well as synergistic effect might be involved here and responsible for such behavior [13]. This may be the explanation for high activity of UAE extract against most of the tested strains. Further
14
research is needed in order to establish the relationship between chemical composition of extracts and their antibacterial activity.
4. Conclusion
The preset study showed that Erica carnea L. possesses a good potential to be used as a source of biologically active compounds. Based on the presented results, it may be noticed that prepared extracts exhibited high antioxidant, cytotoxic and antibacterial activity. Subcritical water extract generally showed the best properties, but other non-conventional techniques demonstrated satisfactory results. Using modern analytical technique, such as HPLC-DAD, presence of the certain phenolic compounds has been confirmed. Analysis showed that amount of the compounds in the extract depends on the applied extraction technique as well as on the experimental conditions. All in all, obtained results in this study should encourage further and deeper investigation of this plant together with the new fields and possibilities for its application.
Acknowledgements
Authors of this study are grateful to Dr Milan Stankoviš, University of Kragujevac, Faculty of Sciences, Institute of Biology, Republic of Serbia, for support in terms of confirmation and deposition of Erica carnea L.
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Table 1. Analytical parameters for 18 phenolic compounds used for HPLC-DAD analysis Calibration (R2) RT* LOD** curve (min) μg/ml Protocatechuic acid y=13307.5x+0.235 0.9988 12.52 0.004 p-Hydroxybenzoic acid y=9934.4x+0.086 0.9998 17.65 0.003 Caffeic acid Y=32241.5-0.154 1.0000 21.19 0.009 Vanillic acid Y=10781.0+0.520 0.9995 22.15 0.003 Chlorogenic acid Y=10491.8-0.525 0.9998 22.25 0.035 Syringic acid Y=11253.6+0.465 0.9999 23.88 0.011 p-Coumaric acid Y=16239.7-0.832 0.9997 24.83 0.042 Ferulic acid Y=24685.7-0.754 0.9998 27.35 0.029 Sinapic acid Y=13332.5+1.062 0.9998 29.11 0.032 Rutin Y=4589.0-0.674 0.9999 29.30 0.052 Luteolin-glycoside Y=13132.5+1.042 0.9998 31.22 0.032 Apigenin-glycoside Y=4289.0-0.674 0.9999 32.21 0.052 Rosmarinic acid Y=5385.2+0.656 0.9998 33.58 0.018 Quercetin Y=10336.2+0.320 0.9996 36.50 0.033 Luteolin Y=15958.6-0.234 0.9998 37.31 0.030 Naringenin Y=14797.5+0.980 1.0000 37.85 0.055 Kaempferol Y=13636.5+0.098 0.9997 38.29 0.029 Apigenin Y=6229.8+1.200 0.9996 40.10 0.045 *RT-retention time, **LOD-limit of detection, ***LOQ-limit of quantification. Compound
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LOQ*** μg/ml 0.013 0.010 0.030 0.010 0.116 0.037 0.140 0.097 0.106 0.173 0.106 0.173 0.060 0.110 0.100 0.183 0.097 0.150
Table 2. Chemical profile of Erica carnea L. extracts obtained by spectrophotometric assays Extract SE
Y (g/100 g DP) 12.10
MAC
15.30
UAE
18.33
MAE
30.65
SCW
42.66
TPC (mg GAE/g) 98.86 ± 0.49 118.14 ± 0.26 127.42 ± 0.87 135.56 ± 0.19 144.12 ± 0.23
TFC (mg RU/g) 19.18 ± 0.37 20.57 ± 0.58 22.55 ± 0.17 23.81 ± 0.60 26.24 ± 0.18
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CT (mg GAE/g) 55.85 ± 0.44 57.19 ± 0.37 60.65 ± 0.54 61.70 ± 0.19 62.53 ± 0.81
GA (mg GAE/g) 17.13 ± 0.94 19.74 ± 0.96 20.19 ± 0.42 23.36 ± 0.77 25.56 ± 0.64
TAC (mg C3G/g) 10.60 ± 0.40 11.86 ± 0.73 12.46 ± 0.19 13.07 ± 0.54 13.13 ± 0.28
Table 3. Polyphenolic profile of Erica carnea L. extracts Compound Protocatechuic acid p-Hydroxybenzoic acid Caffeic acid Vanillic acid Chlorogenic acid Syringic acid p-Coumaric acid Ferulic acid Sinapic acid Rutin Luteolin-glycoside Apigenin-glycoside Rosmarinic acid Quercetin Luteolin Naringenin Kaempferol Apigenin Σ *ND-not determined.
SE ND* 0.102 0.287 0.293 1.217 ND 0.553 0.393 0.238 1.987 ND 0.362 11.890 7.151 0.181 0.306 0.985 1.351 27.296
Content (mg/g) MAC UAE ND ND 0.770 ND ND ND 0.129 ND 0.132 ND ND 0.017 0.108 0.015 0.113 0.022 0.408 0.123 2.159 0.728 0.336 ND ND 0.016 0.746 0.038 1.895 0.149 0.559 0.058 0.055 0.019 0.074 0.032 0.321 0.018 7.805 1.235
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MAE ND 0.121 ND ND ND 0.053 0.044 0.042 0.267 0.908 ND 0.088 0.131 1.000 0.100 0.044 0.105 0.179 2.959
SCW ND 0.148 ND 0.086 ND 0.048 0.054 0.032 0.099 0.397 0.033 0.024 0.036 0.282 0.016 0.013 0.023 0.029 1.320
Table 4. Antioxidant activity of obtained Erica carnea L. extracts Extract SE MAC UAE MAE SCW
TA (µg AA/G) 112.34 ± 0.43 119.92 ± 0.48 134.71 ± 0.29 159.09 ± 0.82 171.32 ± 0.87
ILP50 (µg/mL) 31.35 ± 0.42 30.73 ± 0.08 28.57 ± 0.44 27.56 ± 0.81 21.71 ± 0.45
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OH50 (µg/mL) 33.51 ± 0.88 30.15 ± 0.93 26.83 ± 0.77 24.41 ± 0.81 20.63 ± 0.94
IC50 (µg/mL) 46.68 ± 1.01 37.83 ± 0.98 36.47 ± 0.69 30,60 ± 0.99 23.73 ± 0.53
Table 5. Cytotoxic activity of obtained Erica carnea L. extracts Extract
IC50 (µg/mL) RD cells 32.43 ± 0.46 30.84 ± 0.28 23.54 ± 0.48 19.23 ± 0.79 16.17 ± 0.35 1.4 ± 0.97
Hep2c cells SE 34.26 ± 0.37 MAC 32.28 ± 0.44 UAE 31.70 ± 0.89 MAE 27.54 ± 0.32 SCW 11.54 ± 0.55 cis-DDP* 0.94 ± 0.55 *cis-DDP, cis-diamminedichloroplatinum.
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L2OB cells 33.09 ± 0.65 28.51 ± 0.14 22.63 ± 0.49 20.09 ± 0.60 18.51 ± 0.52 0.72 ± 0.64
Table 6. Pearson’s correlation coefficients among TPC, TFC, CT, GA, TAC, TA, ILP50, OH50, IC50, Hep2c, RD and L2OB Test TPC TFC CT GA TAC TA ILP50 OH50 IC50 Hep2c RD L2OB TPC 1 TFC 0.9650a 1 CT 0.9620a 0.9596a 1 a a GA 0.9594 0.9743 0.9125b 1 TAC 0.9867a 0.9200b 0.9568b 0.9217b 1 b a b a TA 0.9387 0.9794 0.9477 0.9789 0.9073b 1 ILP50 -0.8609c -0.9567b -0.8483c -0.9223b -0.7727d -0.9213b 1 a a a a b a OH50 -0.9819 -0.9971 -0.9677 -0.9781 -0.9463 -0.9765 0.9352b 1 IC50 -0.9781a -0.9749a -0.9156b -0.9916a -0.9395b -0.9558b 0.9186b 0.9829a 1 d b d b d c a c Hep2c -0.7839 -0.8942 -0.7355 -0.8899 -0.6782 -0.8644 0.9783 0.8681 0.8808b 1 RD -0.9476b -0.9798a -0.9860a -0.9448b -0.9256b -0.9838a 0.9018b 0.9787a 0.9320b 0.8119c 1 L2OB -0.9833a -0.9590a -0.9941a -0.9284b -0.9820a -0.9435b 0.8375a 0.9733a 0.9383b 0.7321d 0.9743a 1 a b c d statistically significant at p < 0.01; statistically significant at p < 0.05; statistically significant at p < 0.1, statistically insignificant
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Table 7. Antibacterial activity of obtained Erica carnea L. extracts Strain S. saprophiticus ATCC 15035 S. aureus ATCC 25923 L. ivanovii ATCC 19119 L. inocun ATCC 33090 E. faccalis ATCC 2912 L. monocytogenes ATCC 19112 B. spieizeneii ATCC 6633 E. faccium ATCC 6057 E. coli ATCC 25922 S. enteritidas ATCC 13076 E. aerogenus ATCC 13048 C. freundi ATCC 43864 S. typhimurium ATCC 14028 P. aeroginosa ATCC 27853 P. mirabilis ATCC 35659 *A-Amracin.
SE
MAC
MIC (µg/mL) UAE MAE
31.25
15.82
15.82
31.25
7.81
0.24
250.00
250.00
31.25
62.50
62.50
0.97
31.25
500.00
31.25
250.00
250.00
0.49
125.00
125.00
15.82
125.00
125.00
0.97
62.50
250.00
15.82
250.00
250.00
0.49
125.00
500.00
31.25
62.50
62.50
0.49
125.00
125.00
31.25
250.00
250.00
0.24
250.00
62.50
31.25
500.00
125.00
0.97
7.81
62.50
62.50
125.00
250.00
0.49
125.00
250.00
250.00
250.00
62.50
0.97
62.50
7.81
15.82
500.00
250.00
0.49
125.00
62.50
15.82
250.00
125.00
0.49
31.25
15.82
7.81
500.00
250.00
0.24
125.00
125.00
15.82
62.50
62.50
0.97
250.00
7.81
62.50
250.00
250.00
0.49
28
SCW
A*
Graphical Abstract
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