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Chemicals: acetic acid (A6283), trichloroacetic acid (TCA) (T6399), Trizma base (T4661), sulforhodamine-B (SRB) (230162), aluminum trichloride (237051), sodium acetate (S2889), sulfuric acid 95–97% (10009731), phenol (P1037), hydrochloric acid 37% (H1758), and n-butanol (B7906) were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Solvents: (chloroform, purity ≥ 99.8% (319988), methanol LC-MS, grade ≥ purity 99.9% (34860), water HPLC grade (34877), acetonitrile HPLC grade, purity ≥ 99.9 (34851), acetone ≥ 99.8% (34580), and formic acid LC-MS grade (85178) were purchased from Honeywell (Medisell Nicosia, Cyprus). Analytical standards: ascorbic acid (ST0800102) and Trolox (ST08003) were purchased from Bioquochem (Asturias, Spain). Catechin (43412), mannose (92683), and linalool (62139) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Gallic acid (4993S), chlorogenic acid (4991S), ferulic acid (4753S), ellagic acid (6075), vanillic acid (6113), caffeic acid (6034S), syringic acid (6011), p-coumaric acid (4751S), rosmarinic acid (4957S), 4-hydroxybenzoic acid (6099), protocatechuic acid (6050), 2′-hydroxyflavanone (1180), 7-hydroxyflavanone (1212), 4′-methoxyflavanone (1185), 5-methyxyflavanone (1186), apigenin-7-O-glucoside (1004S), luteolin-7-O-glucoside (1126S), isorhamnetin (120S), quercetin-3-O-rhamnoside (1236S), hyperoside (1027S), myricetin-3-O-galactoside (1355S), kaempferol-3-O-rutinoside (1053), ipriflavone (1328), and naringin (1129S) were purchased from Extrasynthese (Lyon, France). Bovine Serum Albumin (BSA) (23209) was from Thermofisher (Medisell, Nicosia, Cyprus). The bicinchoninic acid (BCA) Protein Assay Kit (23225) was purchased from Thermo Scientific (Waltham, MA, USA). ABTS Assay Kit (KF01002), 2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay Kit (KF01007), and Total Polyphenol Assay Kit (KB03006) were purchased from Bioquochem (Asturias, Spain). Dulbecco’s Modified Eagle’s Medium (DMEM) and phosphate-buffered saline (PBS) were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA); trypsin, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Biosera (Boussens, France).
2.2. Plant Material and Preparation of Plant ExtractPlants of H. rhodopensis were propagated routinely in vitro and adapted in pots under controlled conditions, as previously described [21]. Fully developed leaves from well-hydrated pot plants were detached and air-dried to be used for extraction. For the H. rhodopensis aqueous extract (HAE), air-dried leaves (50 mg) were ground in 1.5 mL distilled water and then boiled for 10 min. The samples were centrifuged at 10,000× g for 10 min. The supernatant was collected and freeze-dried. For the H. rhodopensis ethanol extract (HEE), air-dried leaves (50 mg) were ground in 0.5 mL 70% ethanol. Extraction took place for 48 h at room temperature (RT). The samples were centrifuged at 10,000× g for 10 min and the supernatants were collected. The remaining pellet was subjected to additional extraction for 24 h at RT. After additional centrifugation, the supernatant was collected. Both supernatants were combined and dried at 40 °C by SpeedVac. Both HAE and HEE were stored at −20 °C and dissolved in dimethyl sulfoxide (DMSO) before use. Serial dilutions of dissolved extracts were prepared in culture medium for incubating cells with the extracts. 2.3. Cell LinesThe human cancer cell lines A549 (non-small cell lung adenocarcinoma, ATCC CCL-185), HepG2 (hepatocellular carcinoma, ATCC HB-8065), HT29 (ATCC HTB-38) and Caco-2 (ATCC HTB-37) (colorectal adenocarcinomas), and PC3 (ATCC CRL-1435) and DU145 (ATCC HTB-81) (prostate adenocarcinomas) were obtained from the American Type Culture Collection (Rockville, MD, USA). All cell lines were maintained under sterile conditions at 37 °C in a humidified atmosphere of 5% CO2 and routinely passaged with trypsin. PC3 and DU145 cells were cultured in DMEM-F12 medium whereas the other cell lines were cultured in DMEM. All media were supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL).
2.4. Cell Growth AssayThe cell growth rate was investigated by the sulforhodamine-B (SRB) assay as previously described [22]. The SRB method is a colorimetric assay based on the analysis of cellular protein content that is used for cell density determination [23]. H. rhodopensis extracts were dissolved in DMSO. Cells were incubated with serial dilutions of the extracts’ preparations (DMSO concentration ≤ 0.1% v/v) for either 48 or 72 h, before being stained with SRB. Control cells were incubated with DMSO, whereas unstained samples for each treatment group served as background controls. For the quantification of the optical density, a microplate reader (Enspire, Perkin Elmer, Waltham, MA, USA) was used. At least six replicates for each sample were examined, and each experiment was independently performed at least three times. The percentage of inhibition of cell growth was calculated by the following Formula (1):% growth = mean OD sample/mean OD control × 100
(1)
2.5. Wound Healing AssayThe effect of HEE on the migration potential of HepG2 and A549 cells was examined with the wound healing assay as described previously [24]. Briefly, cells were seeded in 6-well plates and, upon the formation of a confluent monolayer, a 10 µL pipette tip was used to generate a ‘wound’ on the monolayer by scratching it across the well. Loose and detached cells were washed away with PBS and cells were incubated with either DMSO (control) or HEE. Non-toxic HEE concentrations were used (20 or 30 μg/mL for A549 and 25 or 50 μg/mL for HepG2 cells) to prevent cell growth inhibition. Cells were monitored and photographed with a light microscope (Zeiss, Göttingen, Germany) and equipped with a digital camera, up until wound closure. Multiple photographs per time point were captured in three independent experiments. Photographs were analyzed with ImageJ software (NIH, Bethesda, MD, USA) and the average % wound area (% open image area) was calculated. 2.6. Chemical Characterization 2.6.1. Sample ProcessingFor the determination of the total phenolic (TPC), flavonoid (TPC), soluble sugar (TSSC), soluble protein (TSPC), and pigment content, the dried HEE was resolubilized in methanol, whereas HAE was resolubilized in LC-MS grade water. Both reconstituted extracts were filtered through a 0.45 μm membrane filter (Sartorius Stedim biotech, Guttenberg, Germany). The formed solutions were aliquoted and stored at −20 °C protected from light until further use.
2.6.2. Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)The TPC, of reconstituted solutions of HAE and HEE, was analyzed using a commercial polyphenolic quantification assay kit (Folin–Ciocalteu method) (KB03006, Bioquochem, Asturias, Spain) according to the manufacturer’s instructions. The TPC was determined based on the gallic acid calibration curve (linear range: 0–500 μg/mL, y = 0.004496x + 0.01785, R2 > 0.990). The results were expressed as μg of gallic acid equivalents/g of dry extract. The quantification of TFC was performed as it was previously reported with some modifications [25]. Briefly, 40 μL of each solution (HAE or HEE, respectively) was diluted with 120 μL of methanol and mixed with 20 μL of aluminum trichloride (10% aqueous solution) and 20 μL of sodium acetate (0.5 M aqueous solution). The resulting solutions were allowed to stand in the dark at RT for 40 min, and then the absorbance was monitored on a microplate reader (LT4500, Labtech, Heathfield, UK) at 415 nm. The TFC was determined based on the rutin calibration curve (linear range: 0–500 μg/mL, y = 0.0006949x + 0.04410, R2 > 0.993). The results were expressed as μg of rutin equivalents/g of dry extract. 2.6.3. Determination of Total Condensed Tannins Content (TCTC)The determination of TCTC was performed according to a previously published experimental protocol [26]. Briefly, 500 μL of either HAE or HEE reconstituted solution, was diluted with 500 μL of 70% acetone. Then, 3 mL of the n-butanol/hydrochloric acid (37%) mixture (95:5 % v/v) was added, and the resulting solutions were heated at 95 °C for approximately 60 min. Upon completion of the reaction, the solution mixture was allowed to cool at RT, mixed with ammonium iron (III), sulfate (NH4Fe(SO4)2) 2% (w/v), and heated for 2 h at 70 °C. Eventually, the absorbance of the cooled solution mixture was monitored on a microplate reader (LT4500, Labtech, Heathfield, UK) at 550 nm. The TCTC was determined based on a catechin calibration curve (linear range: 10–100 μg/mL, y = 001912x − 0.02036, R2 > 0.993). The results were expressed as μg of catechin equivalents/g of dry extract. 2.6.4. Determination of Total Monoterpenoid Content (TMC)The determination of TMC was performed by adopting a previously reported methodology [27]. Namely, 200 μL of either HAE or HEE reconstituted solution, was mixed thoroughly with 1.5 mL chloroform and allowed to stand for 3 min. Then, 100 μL of concentrated sulfuric acid were added and the suspensions were allowed in the dark for 2 h. The supernatant was decanted, the formed precipitant was taken up in 95% (v/v) methanol, and the absorbance was monitored on a microplate reader (LT4500, Labtech, Heathfield, UK) at 538 nm. The total monoterpenoid content was determined based on a linalool calibration curve (linear range: 0–60 μΜ, y = 0.005074x + 0.003620, R2 > 0.995). The results were expressed as μg of linalool equivalents/g of dry extract. 2.6.5. Determination of Total Soluble Sugar Content (TSSC)The determination of TSSC was performed as it was previously reported with some modifications [25]. Briefly, 150 μL of either HAE or HEE reconstituted solution was dehydrated by adding 150 μL of concentrated sulfuric acid, and the resulting solution mixture was shaken for 30 min at RT. Then, 30 μL of 5% phenol was added, and the final mixture was heated at 90 °C for 5 min. The absorbance of the cooled solutions was monitored on a microplate reader (LT4500, Labtech, Heathfield, UK) at 490 nm. The TSSC was determined based on the sucrose calibration curve (linear range: 0–100 nM, y = 0.01645 + 0.1578, R2 > 0.998). The results were expressed as nmol of mannose equivalents/g of dry extract. 2.6.6. Determination of Total Soluble Protein Content (TSPC)TSPC content was determined by utilizing the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, and the absorbance was monitored at 562 nm using a microplate reader (LT4500, Labtech, Heathfield, UK). The TSPC was calculated based on a standard curve of bovine serum albumin (BSA) (linear range: 0–2 mg/mL, y = 0.6851x + 0.1345, R2 > 0.995). The results were expressed as mg of protein/g of dry extract.
2.6.7. Determination of PigmentsThe total content in chlorophyll-a and -b, lycopene, and β-carotene of either HAE or HEE reconstituted solutions, was determined as previously described [28]. The absorbance was measured sequentially at 453, 505, 645, and 663 nm using a microplate reader (LT4500, Labtech, UK), and the content was calculated using the following Equations (2)–(5):Chlorophyll-a (mg/g of dry extract) = [((0.999 × A663) − (0.0989 × A645))/20]
(2)
Chlorophyll-b (mg/g of dry extract) = [((1.77 × A663) − (0.328 × A663))/20]
(3)
Lycopene (mg/g of dry extract) = [((−0.0458 × A663) + (0.204 × A645) + (0.372 × A505) − (0.0806 × A453))/20]
(4)
β−carotene (mg/g of dry extract) = [((0.216 × A663) − (1.22 × A645) − (0.304 × A505) + (0.452 × A453))/20]
(5)
The results were expressed as μg of pigment (chlorophyll-a or -b or lycopene or β-carotenoid)/g of dry extract.
2.6.8. Determination of VolatilesChemical analysis of polar metabolites contained in an ethanolic sample of H. rhodopensis was performed as previously described [29]. Derivatization by silylation of the metabolites was initiated by the addition of methoxyamine hydrochloride followed by the addition of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) to complete the reaction. The samples were analyzed by gas chromatography/mass spectrometry (GC/MS) on a Thermo Trace Ultra GC gas chromatography system with ISQ MS mass spectrometry and TriPlus RSH (Switzerland). The identification of metabolites and the analysis of mass spectra were performed using databases such as NIST11 and GOLM, while also for the identification of peaks, a comparison was made with standard substances. 2.6.9. Preparation of Standards and SamplesStock solutions of 4-hydroxybenzoic acid, protocatechuic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, caffeic acid, ferulic acid, rosmarinic acid, chlorogenic acid, ellagic acid, 7-hydroxyflavanone, 4′-methoxyflavanone, apigenin-7-O-glucoside, isorhamnetin, quercetin-3-O-rhamnoside, quercetin-3-O-rutinoside (rutin), naringin, kaempferol-3-O-rutinoside, hyperoside, and myricetin-3-O-galactoside were prepared in methanol, luteolin-7-O-glucoside in acetonitrile/water mixture (1:1) and 2′-hydroxyflavanone, 5-methoxyflavanone and ipriflavone in methanol/acetonitrile mixture (1:1) at a concentration of 1000 ppm. Working standard solutions were made by diluting the individual standard stock solutions with ice-cold methanol. Both solutions of HEE and HAE were diluted with ice-cold methanol at a final concentration of 25 ppb. Each solution was kept in the dark and protected from light to minimize the autooxidation of polyphenols and pigments. In addition to this, both stock, standard, and sample solutions were stored at −20 °C before use. All prepared solutions were passed through 0.22 μm membrane filtered prior to UPLC-QqQ-ESI-MS/MS analysis.
2.6.10. Liquid Chromatography (LC) ConditionsFor the detection and quantification of the listed polyphenols, a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA) equipped with an autosampler chamber, two pumps, and a degasser was used. The chromatographic separation was performed on an ACQUITY UPLC BEH C18 (100 × 2.1 mm, particle size: 1.7 μm) column (Waters Corp., Milford, MA, USA), heated at 30 °C and eluted as it was previously reported but with some modification [30]. Briefly, the mobile phase consisted of a solution of acetonitrile (eluent A) and formic acid 0.1% (v/v) (eluent B). A flow rate of 0.3 mL/min was used and the linear gradient conditions applied consisted of 5–100% A (0–4 min), 100–90% A (4.0–4.1 min), 90% A (4.1–5 min), 90–5% A (5–5.1 min), and 5% A (5.1–6 min). The injection volume was 10 μL and the autosampler temperature was set at 4 °C. 2.6.11. MS/MS ConditionsFor the MS/MS experiments, a Xevo tandem (triple) quadrable (QqQ) detector (TQD) mass spectrometer (Waters Corp., Milford, MA, USA) was operated in either positive or negative ionization mode (ESI±). Quantitative analysis was accomplished using selected multiple reaction monitoring (MRM) mode (Figures S1 and S2). The MRM conditions were optimized for each standard by MS manual tuning of each standard prior to sample analysis at a concentration of 1 ppm (Table S1). To acquire maximum signals, the optimized tuning parameters were as follows: capillary voltage: 3.0 kV; cone voltage: 36 V; source temperature: 150 °C; dissolution temperature: 500 °C; source disolving gas flow: 1000 L/h; and gas flow: 20 L/h. High-purity nitrogen gas was used as the drying and nebulizing gas, whereas ultrahigh-purity argon was used as a collision gas. The data acquisition and processing were performed on MassLynx software (version 4.1). 2.7. Determination of Antioxidant Activity 2.7.1. ABTS●+ AssayFor the determination of the ability of the HAE and/or HEE to inhibit the cationic radical of ABTS●+, the ABTS●+ assay kit (KF01002, Bioquochem, Asturias, Spain) was used according to the manufacturer’s instructions. Gallic acid (0–35 μg/mL), Trolox (0–15 μg/mL), and ascorbic acid (0–7 μg/mL) were used as positive controls, whereas the potency of HAE and HEE were examined in a range of concentrations varying from 0 to 80 μg/mL. The results were calculated as % Radical cation inhibition according to Equation (6)% Radical cation inhibition = [1 − (Af/A0)] × 100
(6)
where Af is the absorbance recorded at 517 nm, 5 min after the addition of samples, and A0 is the absorbance of the non-inhibited radical cation at 517 nm and expressed as the half-maximal inhibitory concentration (IC50) in μg/mL. 2.7.2. DPPH● AssayFor the determination of the ability of the HAE and HEE to inhibit the DPPH● radical, the DPPH● assay kit (KF01007, Bioquochem, Asturias, Spain) was used according to the manufacturer’s instructions. Gallic acid (0–3.5 μg/mL), Trolox (0–15 μg/mL), and ascorbic acid (0–70 μg/mL) were used as positive controls, whereas the potency of HAE and HEE were examined in a range of concentrations varying from 0 to 80 μg/mL. The results were calculated as % Radical cation inhibition according to Equation (6) where Af is the absorbance recorded at 715 nm, and A0 is the absorbance of the non-inhibited radical cation at 715 nm and expressed as the half-maximal inhibitory concentration (IC50) in μg/mL.
2.7.3. FRAP AssayFor the determination of the ability of HAE and HEE to reduce the ferrous cationic species, the fast FRAP assay kit (KF01006, Bioquochem, Asturias, Spain) was utilized and performed according to the manufacturer’s recommendation. Gallic acid 0–3.5 μg/mL), Trolox (0–15 μg/mL), and ascorbic acid (0–70 μg/mL) were used as positive controls, whereas the potency of HAE and HEE were examined in a range of concentrations varying from 0 to 80 μg/mL. The results were calculated as mmols of Fe2+/g of dry extract and expressed as the half-maximal inhibitory concentration (IC50) in μg/mL.
2.8. Statistical AnalysisData are presented as mean ± SD and are representative of at least three independent experiments. Sigma Plot v.11 software (Systat Software Inc., San José, CA, USA) was used for statistical analyses and for generating the graphs unless stated otherwise. For statistical comparisons, Student’s t-test was used for comparing two groups, whereas, for multiple group comparisons, one-way ANOVA was performed. Differences between groups were considered significant when p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001). EC50 values (Efficient Concentration; the concentration that induces a 50% cell growth inhibition) were calculated from the respective dose-response curves by regression analysis using a four-parameter logistic curve with the Sigma Plot v. 11 Software v.10.
4. DiscussionThe use of various plant extracts as a source of medicinal agents (pharmaceuticals), remedies, cosmetics, and food additives continues to be an attractive alternative for the industry. For centuries, the local population has gained knowledge and experience in using various plant extracts in folk medicine, local kitchen (cuisine), and religious rituals. This tradition continues today when the use of the enormous local plant biodiversity is supported with extensive scientific research. Working together in RESBIOS, a Horizon 2020 project dedicated to incorporating the Responsible Research and Innovation (RRI) principles [32] in science, the groups from AgroΒioΙnstitute in Bulgaria and Democritus University of Thrace in Greece, found a scientific field of joint interest—the potential use of plant extracts of local interest in human cancer treatment. Moreover, we chose to focus our research on the locally endemic species H. rhodopensis. H. rhodopensis is a rare species, endemic to certain parts of Bulgaria and Greece. Fostering a responsible research approach in respecting bioconservation in our study, we utilized only extracts from biotechnologically obtained plants, since the species is protected. It is confirmed that in vitro-developed plants that are adapted in pots and grown under controlled conditions are highly similar to those collected from nature [21].H. rhodopensis belongs to the small group of so-called resurrection plants known for the extreme desiccation tolerance of their vegetative parts. Being a subject of numerous extensive studies, it has been concluded that H. rhodopensis exerts significant antioxidant properties as other resurrection species do, to ensure desiccation tolerance. The significant antioxidant activity that H. rhodopensis extracts have, increased the interest in studies in medicinal and human well-being fields [6]. However, it is difficult to find any ethnobotanical-based evidence indicating the use of H. rhodopensis by the local population in the past. In the last 20–30 years though, the interest in various potential biological activities of these extracts raised enormously. Various extraction techniques and solvents have been used but as a rule, the studies on the biological activity of the extracts obtained were not accompanied by any chemical characterization of their content. Most studies so far have been performed on methanol extracts [3,20,33,34]. In general, aqueous and ethanol extracts are considered less toxic and more suitable for in vitro bioactivity studies. In the present study, we used aqueous and ethanol extracts from air-dried leaves of H. rhodopensis plants in order to perform a comparative study on their antiproliferative potential against a panel of cancer cell lines and further focused on the ethanolic extract of the plant that demonstrated stronger bioactivity.The comparison of aqueous (HAE) and ethanol (HEE) extracts showed that both of them exhibited a dose- and time-dependent growth inhibitory activity. However, HAE induced weaker antiproliferative effects compared to HEE (Figure 1 and Figure 2, Table 1 and Table 2). Similar results reporting weak cytotoxicity for the aqueous extracts have been described earlier [15] and in this respect, it was decided to focus on HEE. Methanol extracts promoted H2O2-induced apoptosis in prostate cancer (PC3) cells and exerted a protective effect against the non-cancerous (HEK293) cell line under the same experimental conditions [11].In our study, the antiproliferative activity of the extracts was assessed using a platform of six different human cancer cell lines. We found that the different types of cancer cells had differential sensitivity to either HAE or HΕE with the more sensitive being the androgen-independent prostate (DU145), the hepatocellular carcinoma (HepG2), and the non-small cell lung adenocarcinoma (A459) cells. HEE also significantly affected the growth of the colon adenocarcinoma (Caco-2 and HT29) cell lines whereas it did not appear to have any effect on the growth of prostate (PC3 and DU145) cancer cells. The differential bioactivity of HEE, on the two prostate cancer cell lines tested, could be associated with their differences in expressing prostate-specific antigen (PSA) [35]. Moreover, DU145 cells have moderate metastatic potential compared to PC3 cells which have a high metastatic potential [36]. Noteworthy, contrary to cancer cells, normal cells were not equally sensitive to HEE as shown in Figure S4. An EC50 value of 221 ± 53 μg/mL was estimated for HaCaT keratinocytes at 72 h, while the highest EC50 value observed for the cancer cells lines examined, was 168 ± 45 μg/mL for Caco-2 cells. The lower ≈30% EC50 value in cancer cells as compared to normal cells, indicates that cancer cells, under the experimental conditions employed, are more susceptible to the growth inhibitory effects exerted by HEE compared to the normal cell line tested. Finally, a significant antimigratory potential of HEE was observed against HepG2 and A549 cells. To our knowledge, this is the first study describing the antiproliferative and antimigratory effects of H. rhodopensis extracts that may be of significant value for further exploring their anticancer potential.To this end, our data prompted us to further characterize the chemical composition of both HAE and HEE. HEE was found to contain both flavonoids and phenolic compounds in abundance with the latter being more prominent. On the contrary, the levels of monoterpenoids and condensed tannins were present at low levels. Other significant molecules included soluble proteins and sugars both of which were present at high and low levels, respectively. In addition, the HEE extract contained high amounts of pigments (chlorophyll-a, -b, lycopene, and β-carotene) with chlorophyll-a being present at the highest concentration levels among the other pigments. On the other hand, analysis of HAE indicates the presence of polyphenolic compounds and pigments, amounting to much less when compared to HEE. In addition to this, it was observed that HAE contains a higher amount of soluble proteins and sugars when compared to HEE. The results of our study are in accordance with the main type of bioactive molecules that have been identified in various H. rhodopensis extracts, as summarized in Georgiev et al. [2], and include flavonoids, phenolic acids, fatty acids, phytosterols, polysaccharides, carotenoids, and other lipid-soluble constituents.Analysis of the polyphenolic profile (including phenolic acids, flavanones, flavones, flavanols, and isoflavones) of the HEE extract revealed that protocatechuic acid and caffeic acid were the most abundant among the phenolic acids whereas ipriflavone and quercetin-3-O-rutinoside were also detected at higher concentrations amongst the flavonoids. We also found other noteworthy phenolic acids in abundance including ellagic acid, gallic acid, ferulic acid, syringic acid, rosmarinic acid, 4-hydroxybenzoic acid, and chlorogenic acid. In the case of flavonoids, quercetin-3-O-rutinoside, kaempherol-3-O-rutinoside, isorhamnetin, luteolin-7-O-glucoside, hyperoside, 7 hydroxyflavanone, apigenin-7-O-glucoside, naringin, 2′-hydroxyflavone, 4′-methoxyflavone, and quercetin-3-O-rhamnoside were all shown to be present. A large number of flavonoids and phenolics were identified earlier by HPLC analysis in a 70% ethanol extract of H. rhodopensis (collected from their natural habitat), prepared by conventional heat reflux extraction, where sinapic, ferulic, and p-coumaric acids were the most abundant amongst the phenolic acids while luteolin followed by hesperidin were the most predominant among the flavonoids [37]. In another study, HPLC analysis on ultrasound-based H. rhodopensis ethanol extract revealed a large number of phenolic acids and flavonoids to be present, with the most abundant phenolic acids being ferulic and sinapic acids, while the major flavonoids were luteolin, hesperidin, and kaempferol [19]. Sinapic acid was not included in our authentic standards in this study and so there is no information about its content in the HEE fraction however, no traceable p-coumaric acid levels were detected. Syringic acid was the most abundant among the five free phenolic acids when GC-MS analyses were performed with methanol extracts [20]. It is obvious, that the differences in the nomenclature of metabolites identified are related to the origin of the plant material used and the extraction methodologies followed. Phenolic compounds, on the other hand, are well known to play a significant role in the orchestrated response of plants under stress conditions and their recruitment, at high levels, is well documented in important physiological functions of the resurrection plants [38].As it was anticipated, based on the high phenolic and flavonoid content of the HEE, a remarkable antioxidant capacity was shown as measured by three independent cell free-based methodologies including the DPPH● radical scavenging activity, ABTS●+ cation decolorization activity, and FRAP assays. Moreover, such activity was comparable to other potent antioxidants like Trolox as well as gallic and ascorbic acids. High in vitro antioxidant activity for H. rhodopensis extracts have also been reported by other studies using various assays [2]. On the other hand, the antioxidant evaluation of HAE demonstrated some radical scavenging potency. However, the marked capacity was considerably weaker compared to the one noticed for HEE. This difference can be attributed to the low content of ROS scavenger molecules (e.g., low content in polyphenolic acid, flavanones chlorophylls, etc.). The observed antioxidant activity of HEE is promising and indicative of potential cytoprotective properties. H. rhodopensis as a source of antioxidant compounds should be further explored by utilizing physiologically relevant cell-based models and in vivo studies to demonstrate its possible health-promoting effects as well as potential biomedical applications.
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