Abstract
The multi-component fingerprint and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products. We investigated the quantitative content of biologically active compounds (anthocyanins and chlorogenic acid) of microwave-assisted blueberry extracts from 14 different Italian cultivars, using validated high-performance liquid chromatography-photodiode array detector (HPLC-PDA) method and routinely instrument configuration. The carbonic anhydrase (CA, EC 4.2.1.1) inhibition profiles against several pharmacologically relevant CA isoforms of blueberry extracts and some bioactive compounds were also investigated. The various cultivars showed a highly variable content in anthocyanins and chlorogenic acid, and their CA inhibitory effects were also highly variable. Overall these data prove that antioxidant natural products found in blueberries may be useful for designing pharmacological agents in which various CAs are involved, e.g., antiobesity, antitumor, or anticonvulsants agents.
Introduction
The multi-component pattern and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products available on the market. The biologically active compounds and their quantitative analysis provide the complete characterization of such final products. Furthermore, due to the wide occurrence of these nutrients, valuable effects related to their intake were observed on human health. In this context, studies investigating this topic showed the pivotal role played by nutraceutical research for new drug development processesCitation1.
These results enhanced the attention, especially from Pharmaceutical and Food Industries, on naturally occurring compounds which may be devoid of the classical drawbacks related to adverse effects of synthetic derivatives, which could be considered not merely as a nutrients source but also as supplements helping conventional therapies against various diseasesCitation2. In particular, recently, Supuran et al.Citation3–8 reported that some phenolic compounds among plant secondary metabolites were active against well-established pharmacological targets such as human carbonic anhydrases (hCAs), in addition to their well-known activities such as antioxidant, anti-inflammatory, and anti-carcinogenic compoundsCitation9,Citation10. In the last years, secondary plant metabolitesCitation11–14 and specifically phenols have been positively used in quality control on different plant-derived products belonging to several chemical classes.
Pursing our research on blueberry, after having reported an untargeted NMR metabolic fingerprint of their extracts and put in evidence the presence of several anthocyanins in addition to chlorogenic acid and fatty acidsCitation15,Citation16, we aimed at characterizing the multi-component fingerprint of this natural product highlighting the differences/similarities among 14 different cultivars from the same area (Latium district, Italy) in terms of anthocyanins and chlorogenic acid content after microwave-assisted extraction (MAE) (). Finally, taking into account the circumstantial literature, we endeavored to undertake an in vitro assessment of these blueberry extracts to determine their inhibitory effects against four selected human carbonic anhydrase (hCA) isoforms (hCA I, II, VA, and VII)Citation17. In this contest, blueberries are well known for their complex phenolics chemical profilesCitation18,Citation19, but up to date, no research was carried out in order to clarify their possible inhibition activity on hCA isoforms, and particularly supporting these findings using a specific multi-component pattern.
Figure 1. Chemical structures of the reported analytes.
A validated analytical approach combined with biological methodologies, able to evaluate the inhibitory activities of plant-derived extracts, is thus fundamental for this type of research. Furthermore, the different extraction techniques could modify the multi-component pattern and the corresponding biological activityCitation20,Citation21. For this reason, we carried out an MAE applying our optimized procedure of Bligh–Dyer methodCitation15 with a three solvents system (water–methanol–chloroform) in order to obtain two different extracts (hydroalcoholic and organic) enriched in specific components.
In continuation to our studies on natural products analyses and characterizationCitation22–30, and instrument configurationsCitation31–33, we report herein, for the first time, a targeted multi-component pattern and biological activity against four selected human carbonic anhydrase isoforms (I, II, VA, and VII) of 14 different blueberry cultivars after MAE in order to evaluate innovative sources of natural ingredients for bioactive formulations.
Materials and methods
Samples
Blueberries were hand harvested (in 2013) in an experimental field located in Ciampino, Lazio, Italy (CREA). Commercial standards of malvidin-3-O-glucoside chloride (oenin, >95%), cyanidin-3-O-rutinoside chloride (keracyanin, >96%), cyanidin-3-O-galactoside chloride (ideain, >97%), and delphinidin-3-O-glucoside chloride (myrtillin, >95%) were purchased from Extrasynthese (Lyon, France). Malvidin-3-O-galactoside chloride (primulin, >95%) and delphinidin-3-O-galactoside chloride (empetrin, >95%) were supplied by PhytoLab (Vestenbergsgreuth, Germany). Chlorogenic acid (>95%) was purchased from Sigma-Aldrich (Milano, Italy). Commercial standard of 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (>99%) was supplied by Avanti Polar Lipids (Alabaster, AL) as an internal standard to validate the efficiency of the extraction recovery.
Sample extraction
MAE was performed using an automatic Biotage InitiatorTM 2.0 (Uppsala, Sweden), 2.45 GHz high-frequency microwaves, with a power range of 0–300 W. An IR sensor probe controlled strictly the internal vial temperature. Few berries of each blueberry cultivar (about 2 g) were placed in liquid nitrogen and finely powdered in a mortar with a pestle followed by the addition of methanol and chloroform mixture (2:1, v:v) at 4 °C (3 mL). The sample was transferred in a 10 mL sealed vessel suitable for an automatic single-mode microwave reactor and 1 mL of chloroform and 1.2 mL of distilled water were added to the sample to form an emulsion. MAE was carried out heating by microwave irradiation for 20 min at 40 °C, and then cooling with pressurized air. The homogenate was then centrifuged (10 000 rpm for 20 min at 4 °C). The upper (hydroalcoholic) and lower (organic) phases were carefully separated. The pellets were re-extracted using half of the solvent volumes (in the same conditions described above) and the separated fractions were pooled. Both fractions were dried under an N2 flow at room temperature until the solvent was completely evaporated. The dried phases were stored at –20 °C until the HPLC analysis.
HPLC-specific multi-component pattern analysis
The specific multi-component pattern for the different blueberry cultivars was analyzed using validated HPLC-PDA method (see Supplementary material, Section S(0).1 for validation parameters) in a gradient elution mode using water and acetonitrile (both 0.1% trifluoroacetic acid) at 95%:5% as starting gradient percentages. The pure chemical standards were used for the quantification of each phenolic compound in external-matrix matched calibration method.
The validated procedure allows the accurate (precise and true) quantification within the linearity range (1–100 μg/mL) for both hydroalcoholic and organic extracts and the reported values are expressed as μg/mg of dry extract (as μg/mL and as percentage are reported in Supplementary material, Section S.2).
Biological evaluation
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity. Phenol red (0.2 mM) has been used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5, for α-CAs) as a buffer and 20 mM NaClO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10–100 sCitation34. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor/sample, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1 mM) or extract sample (1 mg/mL) were prepared in distilled-deionized water and dilutions were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. The inhibition data were obtained by non-linear least-squares methods using PRISM 3 and represent the average from at least three different determinationsCitation35,Citation36. All recombinant CA isoforms were obtained in-house as previously reportedCitation37,Citation38.
Results and discussion
HPLC analysis of the specific multi-component pattern of blueberry extracts
Using the validated HPLC-PDA method, the multi-component fingerprint and the specific capability for the three different solvents to extract specific bioactive compounds have been investigated, as reported in and for all hydroalcoholic and organic extracts, respectively, of each cultivar. Generally, the hydroalcoholic extracts are richer in terms of bioactive compounds with respect to the organic extracts. The more representative compounds were delphinidin-3-O-galactoside (mean value among the cultivars = 9.74 μg/mg of dry extract), malvidin-3-O-galactoside (mean value among the cultivars = 3.85 μg/mg of dry extract), cyanidin-3-O-rutinoside (mean value among the cultivars = 3.80 μg/mg of dry extract), and chlorogenic acid (mean value among the cultivars = 3.98 μg/mg of dry extract), whereas the richer blueberry hydroalcoholic extracts are those belonging to Spring Wind, Gulf Coast, Chandler, and Blue Crop cultivars with a total content of 57.34, 44.52, 43.46, and 40.14 μg/mg of dry extract, respectively. Each cultivar has a characteristic content of anthocyanins/chlorogenic acid.
Table 1. Multi-component pattern for hydroalcoholic extracts of fourteen Italian cultivars expressed as μg/mg of dry extract.
Table 2. Multi-component pattern for organic extracts of fourteen Italian cultivars expressed as μg/mg of dry extract.
Conversely and as expected, organic extracts showed a very poor chemical profile for these bioactive compounds. The bioactive molecules quantified in these extracts were small amounts of malvidin-3-O-galactoside (mean value among the cultivars = 0.96 μg/mg of dry extract), malvidin-3-O-glucoside chloride (mean value among the cultivars = 1.30 μg/mg of dry extract), and chlorogenic acid (mean value among the cultivars = 2.26 μg/mg of dry extract). Chlorogenic acid was quantified especially in Gulf Coast cultivar and, to a less extent, in Spartan, Palmetto, Snow Chaser, Misty, and Emerald.
These findings are in good accordance with previously reported studies on the characterization of blueberry cultivars from other countriesCitation39,Citation40, even if the extraction technique employed in this work is different. The results carried out with our optimized HPLC-PDA procedure were also in agreement with those observed by NMR metabolic fingerprint previously reported by usCitation15.
Carbonic anhydrase inhibition assays
Both the hydroalcoholic and organic extracts of the 14 Italian blueberry cultivars were further assayed against four selected hCA isoforms involved in the physiopathology of important diseases. These isoforms catalyze the same enzymatic process (interconversion between CO2 and bicarbonate), but they differ by kinetics, tissue, and cellular localization as well as sensibility to inhibitors. hCA I and II are the main targets for the treatment of glaucoma and hypertension, since their inhibition leads to blockade of the ciliary process of bicarbonate secretion within the aqueous humor of the eye. Reduction of the rate of bicarbonate and aqueous humor secretion leads to a diminished intra-ocular pressureCitation41,Citation42. hCA VA (and B) are the only CA isoform found in mitochondria, being involved, probably assisted by the cytosolic isozyme hCA II, in ureagenesis, gluconeogenesis, and lipogenesis. hCA VA ensures the presence of bicarbonate in several biosynthetic processes providing enough substrate for de novo lipogenesis. Several studies have provided evidence that its inhibition can reduce lipogenesis in adypocytes in cell cultureCitation43,Citation44. Lastly, hCA VII high expression is observed throughout the cortex, hippocampus, and thalamus and increased intrapyramidally promoting excitatory response (epilepsy) evoked by a GABAergic activityCitation45.
Inhibition data (IC50 for all the 28 extracts and Ki values for the two representative anthocyanins and chlorogenic acid) are collected in and . Delphinidin-3-O-galactoside and cyanidin-3-O-rutinoside were more selective but weaker inhibitors of hCA VA, in the micromolar range when compared with the reference drug acetazolamide. No inhibition has been demonstrated for hCA I, II, and VII. Chlorogenic acid was not active or a weak inhibitor of hCA I, II, and VII, but it displayed a strong inhibitory effect against hCA VA isoform. Conversely and interestingly, some of the hydroalcoholic extracts displayed strong inhibition of the investigated isoforms, thus highlighting that the pharmacological activity must be attributed to other bioactive compounds. As a matter of fact, some of the organic extracts, less rich in anthocyanins but richer in long chain fatty acidsCitation15, showed a particular biological profile. These results were also in agreement with similar reports dealing with anthocyanins-enriched extracts of Turkish sweet cherry cultivarsCitation46. The composition of the extracts seems to be too much complex in order to unravel the relationship between the biological activity and the specific anthocyanins/chlorogenic acid content, but our findings demonstrate a putative pharmacological activity of this natural product strictly correlated to the characteristics of the cultivar more than to their anthocyanins content (at least regarding the most abundant components such as delphinidin-3-O-galactoside, cyanidin-3-O-rutinoside and chlorogenic acid).
Table 3. Inhibition data of human CA isoforms hCA I, II, VA, and VII for reported Blueberry cultivar extracts by a stopped flow CO2 hydrase assay. O, organic extract; A, hydroalcoholic extract.
Table 4. Inhibition data of human CA isoforms hCA I, II, VA, and VII of the two representative anthocyanins, chlorogenic acid and the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.
Considering the extremely fine pH dependency of anthocyanin structure stability, we tested both the extracts () and the single chemical standards (), quantified by HPLC-PDA procedure, strictly at the same pH (7.5) as reported in section Biological evaluation. Under the same experimental conditions, we could disregard the impact of anthocyanin-hindered species (cation, quinoide, and chalcone) on the biological activities on different CA isoforms.
Conclusion
In the present paper, we report for the first time a targeted multi-component pattern analysis by MAE-HPLC-PDA and biological activity of 14 different blueberry cultivars, particularly investigated for their inhibition of carbonic anhydrase isoforms. To the best of our knowledge, these findings have not been reported before and in particular with the aim to estimate these extracts as valuable natural-derived inhibitor sources for carbonic anhydrase isoforms. Founded on the obtained results, the analyzed species exhibited notable biological properties, which should be further explored.
Our new HPLC-PDA method for the determination of these metabolites after microwave-assisted extraction fulfils the acceptance criteria generally established for bio-analytical assays when they are applied in pharmaceutical analysis. In the explored range, this method is accurate (precision and trueness), selective, and sensitive enough to allow the analysis in both the extracts. The analysis can be carried out by means of a relatively simple procedure, with a reduction of analytical variability and sample handling time. In addition, the microwave-assisted procedure can be seen as an alternative to the well-established Bligh–Dyer extraction.
Declaration of interest
The authors report that they have no conflict of interest. This work was supported by “e-ALIERB” Project (Regione Lazio LR13/2008–Dipartimento di Chimica e Tecnologie del Farmaco).
Supplementary material available online
IENZ_1214951_Supplementary_Material.pdf
Download PDF (273.4 KB)References
- Santini A, Novellino E. Nutraceuticals: beyond the diet before the drugs. Curr Bioact Comp 2014;10:1–12
- Shahidi F. Functional foods: their role in health promotion and disease prevention. J Food Sci 2004;69:R146–9
- Karioti A, Carta F, Supuran CT. An update on natural products with carbonic anhydrase inhibitory activity. Curr Pharm Des 2016;22:1570–91
- Gülçin İ, Scozzafava A, Supuran CT, et al. The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase, butyrylcholinesterase, glutathione S-transferase, lactoperoxidase, and carbonic anhydrase isoenzymes I, II, IX, and XII. J Enzyme Inhib Med Chem 2015. [Epub ahead of print]. doi:10.3109/14756366.2015.1094470
- Inci Gul H, Yamali C, Tugce Yasa A, et al. Carbonic anhydrase inhibition and cytotoxicity studies of Mannich base derivatives of thymol. J Enzyme Inhib Med Chem 2016. [Epub ahead of print]. doi:10.3109/14756366.2016.1140755
- Gülçin İ, Scozzafava A, Supuran CT, et al. Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase, lactoperoxidase, acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase isoenzymes. J Enzyme Inhib Med Chem 2016. [Epub ahead of print]. doi:10.3109/14756366.2015.1135914
- Gocer H, Topal F, Topal M, et al. Acetylcholinesterase and carbonic anhydrase isoenzymes I and II inhibition profiles of taxifolin. J Enzyme Inhib Med Chem 2016;31:441–7
- Gidaro MC, Alcaro F, Carradori S, et al. Eriocitrin and apigenin as new carbonic anhydrase VA inhibitors, from a virtual screening of Calabrian natural products. Planta Med 2015;81:533–40
- Zengin G, Ceylan R, Guler GO, et al. Enzyme inhibitory effect and antioxidant properties of Astragalus lagurus extracts. Curr Enzyme Inhib 2016;12:276–81
- Manganaris GA, Goulas V, Vicente AR, Terry LA. Berry antioxidants: small fruits providing large benefits. J Sci Food Agric 2014;94:825–33
- Kosalec I, Kremer D, Locatelli M, et al. Anthraquinone profile, antioxidant and antimicrobial activity of bark extracts of Rhamnus alaternus, R. fallax, R. intermedia and R. pumila. Food Chem 2013;136:335–41
- Kremer D, Kosalec I, Locatelli M, et al. Anthraquinone profiles, antioxidant and antimicrobial properties of Frangula rupestris (Scop.) Schur and F. alnus Mill. Bark Food Chem 2012;131:1174–80
- Mollica A, Locatelli M, Stefanucci A, Pinnen F. Synthesis and bioactivity of secondary metabolites from marine sponges containing dibrominated indolic systems. Molecules 2012;17:6083–99
- Zengin G, Menghini L, Malatesta L, et al. Comparative study of biological activities and chemical fingerprint of two wild Turkish species: Asphodeline anatolica and Potentilla speciosa. J Enzyme Inhib Med Chem 2016. [Epub ahead of print]. doi:10.1080/14756366.2016.1178247
- Capitani D, Sobolev AP, Delfini M, et al. NMR methodologies in the analysis of blueberries. Electrophoresis 2014;35:1615–26
- Sobolev AP, Mannina L, Proietti N, et al. Untargeted NMR-based methodology in the study of fruit metabolites. Molecules 2015;20:4088–108
- Supuran CT. Carbonic anhydrases: from biomedical applications of the inhibitors and activators to biotechnological use for CO(2) capture. J Enzyme Inhib Med Chem 2013;28:229–30
- Barnes JS, Nguyen HP, Shen S, Schug KA. General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography-electrospray ionization-ion trap-time of flight-mass spectrometry. J Chromatogr A 2009;1216:4728–35
- Giovanelli G, Brambilla A, Rizzolo A, Sinelli N. Effects of blanching pretreatment and sugar composition of the osmotic solution on physicochemical, morphological and antioxidant characteristics of osmodehydrated blueberries (Vaccinium corymbosum L.). Food Res Int 2012;49:263–71
- De Monte C, Carradori S, Granese A, et al. Modern extraction techniques and their impact on the pharmacological profile of Serenoa repens extracts for the treatment of lower urinary tract symptoms. BMC Urol 2014;14:63
- Carradori S, Mannina L, De Cosmi F, et al. Optimization of the microwave-assisted extraction of Azadirachta indica (Neem) leaves using NMR-based metabolic fingerprinting. Curr Bioact Comp 2015;11:142–5
- Locatelli M, Tammaro F, Menghini L, et al. Anthraquinone profile and chemical fingerprint of Rhamnus saxatilis L. from Italy. Phytochem Lett 2009;2:223–6
- Genovese S, Tammaro F, Menghini L, et al. Comparison of three different extraction methods and HPLC determination of the anthraquinones Aloe-emodine, Emodine, Rheine, Chrysophanol and Physcione in the bark of Rhamnus alpinus L. (Rhamnaceae). Phytochem Anal 2010;21:261–7
- Epifano F, Genovese S, Kremer D, et al. Re-investigation of the anthraquinone pool of Rhamnus spp.: madagascin from the fruits of Rhamnus cathartica and R. intermedia. Nat Prod Commun 2012;7:1029–32
- Genovese S, Epifano F, Curini M, et al. Screening for oxyprenylated anthraquinones in Mediterranean Rhamnus species. Biochem Syst Ecol 2012;43:125–7
- Celia C, Trapasso E, Locatelli M, et al. Anticancer activity of liposomal bergamot essential oil (BEO) on human neuroblastoma cells. Colloids Surf B Biointerfaces 2013;112:548–53
- Epifano F, Fiorito S, Carlucci G, et al. Phytochemistry and pharmacognosy of naturally occurring prenyloxyanthraquinones. Curr Drug Targets 2013;14:959–63
- Melucci D, Fedi S, Locatelli M, et al. Application of pyrolysis-gas chromatography-mass spectrometry and multivariate analysis to study bacteria and fungi in biofilms used for bioremediation. Curr Drug Targets 2013;14:1023–33
- Genovese S, Epifano F, Carlucci G, et al. HPLC analysis of 4′-geranyloxyferulic and boropinic acids in grapefruits of different geographical origin. Phytochem Lett 2014;8:190–2
- Epifano F, Fiorito S, Locatelli M, et al. Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F. Nat Prod Res 2015;29:180–4
- Locatelli M. Anthraquinones: analytical techniques as a novel tool to investigate on the triggering of biological targets. Curr Drug Targets 2011;12:366–80
- Locatelli M, Genovese S, Carlucci G, et al. Development and application of high-performance liquid chromatography for the study of two new oxyprenylated anthraquinones produced by Rhamnus species. J Chromatogr A 2012;1225:113–20
- Zaza S, Lucini SM, Sciascia F, et al. Recent advances in the separation and determination of impurities in pharmaceutical products. Instrum Sci Technol 2015;43:182–96
- Khalifah RG. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem 1971;246:2561–73
- Carta F, Osman SM, Vullo D, et al. Poly(amidoamine) dendrimers with carbonic anhydrase inhibitory activity and antiglaucoma action. J Med Chem 2015;58:4039–45
- Touisni N, Maresca A, McDonald PC, et al. Glycosyl coumarin carbonic anhydrase IX and XII inhibitors strongly attenuate the growth of primary breast tumors. J Med Chem 2011;54:8271–7
- Truppo E, Supuran CT, Sandomenico A, et al. Carbonic anhydrase VII is S-glutathionylated without loss of catalytic activity and affinity for sulfonamide inhibitors. Bioorg Med Chem Lett 2012;22:1560–4
- Di Fiore A, Truppo E, Supuran CT, et al. Crystal structure of the C183S/C217S mutant of human CA VII in complex with acetazolamide. Bioorg Med Chem Lett 2010;20:5023–6
- Li D, Meng X, Li B. Profiling of anthocyanins from blueberries produced in China using HPLC-DAD-MS and exploratory analysis by principal component analysis. J Food Comp Anal 2016;47:1–7
- Wang L-J, Wu J, Wang H-X, et al. Composition of phenolic compounds and antioxidant activity in the leaves of blueberry cultivars. J Funct Foods 2015;16:295–304
- Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81
- Supuran CT. How many carbonic anhydrase inhibition mechanisms exist? J Enzyme Inhib Med Chem 2016;31:345–60
- Hazen SA, Waheed A, Sly WS, et al. Differentiation-dependent expression of CA V and the role of carbonic anhydrase isozymes in pyruvate carboxylation in adipocytes. FASEB J 1996;10:481–90
- Supuran CT. Carbonic anhydrase inhibitors in the treatment and prophylaxis of obesity. Exp Opin Ther Patents 2003;13:1545–50
- Alterio V, Di Fiore A, D’Ambrosio K, et al. Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? Chem Rev 2012;112:4421–68
- Demir T, Gunesli Z, Sonmez F, et al. Inhibition of carbonic anhydrase I and II with total anthocyanins extracted from sweet cherry cultivars. Environ Eng Manag J 2015;14:935–41