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Anti-inflammatory and angiogenic activity of polysaccharide extract obtained from Tibetan kefir
Microvascular Research 108 (2016) 29–33
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Anti-inflammatory and angiogenic activity of polysaccharide extract obtained from Tibetan kefir Maria Rosa Machado Prado a,⁎, Christian Boller a, Rosiane Guetter Mello Zibetti a,c, Daiany de Souza c, Luciana Lopes Pedroso b, Carlos Ricardo Soccol d a
Faculdades Pequeno Príncipe, Curitiba, PR, Brazil Instituto de Tecnologia do Paraná, Paraná, Curitiba, PR, Brazil Instituto de Pesquisa Pelé Pequeno Príncipe de Pesquisa, Curitiba, PR, Brazil d Universidade Federal do Paraná, Curitiba, PR, Brazil b c
a r t i c l e
i n f o
Article history: Received 19 April 2016 Revised 4 July 2016 Accepted 10 July 2016 Available online 11 July 2016 Keywords: Tibetan kefir Angiogenic activity Anti-inflammatory activity Hyaluronidase
a b s t r a c t The search for new bioactive molecules is a driving force for research pharmaceutical industries, especially those molecules obtained from fermentation. The molecules possessing angiogenic and anti-inflammatory attributes have attracted attention and are the focus of this study. Angiogenic activity from kefir polysaccharide extract, via chorioallantoic membrane assay, exhibited a pro-angiogenic effect compared with vascular endothelial factor (pro-angiogenic) and hydrocortisone (anti-angiogenic) activity as standards with an EC50 of 192 ng/mL. In terms of anti-inflammatory activity determined via hyaluronidase enzyme assay, kefir polysaccharide extract inhibited the enzyme with a minimal activity of 2.08 mg/mL and a maximum activity of 2.57 mg/mL. For pharmaceutical purposes, kefir polysaccharide extract is considered to be safe because it does not inhibit VERO cells in cytotoxicity assays. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Many studies have been conducted with different molecules, extracts and products obtained by fermentation in order to reveal and confirm their biological activity and use in the development of new pharmaceutical, cosmetic and nutraceutical products. Kefir is a fermented milk product with an acidic taste and a creamy consistency that originated in the Balkans, Eastern Europe and the Caucasus (Fontán et al., 2006; Serafini et al., 2014). Kefir has beneficial effects on human health thanks to its antimicrobial (Anselmo et al., 2010), immunoregulatory (Hong et al., 2009), antiallergenic (WeiSheng et al., 2010), antitumoral (Gao et al., 2013), anti-inflammatory (Diniz et al., 2003), antidiabetic (Young-In et al., 2006) and antimutagenic (Guzel-Seydim et al., 2006) activities. The bioactivities of the kefir are attributed to the microorganisms and the substances within it, such as exopolysaccharides produced during the fermentation process of kefir grains. Polysaccharides obtained from different sources, such as plants, animals and microorganisms, have been studied for possessing different biological activities: antibiotic, antioxidant, anticoagulant, anti-mutant,
⁎ Corresponding author at: Faculdades Pequeno Príncipe, Avenida Iguaçu 333, Curitiba, PR 80230-020, Brazil. E-mail address: [email protected] (M.R.M. Prado).
http://dx.doi.org/10.1016/j.mvr.2016.07.004 0026-2862/© 2016 Elsevier Inc. All rights reserved.
immuno-stimulation and anti-cancer (Yu et al., 2009; Wijesekara et al., 2011; Zong et al., 2012). However, polysaccharides derived from microorganisms—especially bacteria—are still being assessed for these activities and others (e.g., angiogenesis and anti-inflammatory). The first studies on angiogenesis were proposed by Folkman (1971) who applied the concept of vascular bed control (i.e., stimulation or inhibition of new vessels) in an attempt to elucidate this process and also as an alternative treatment for various medical conditions (Folkman, 1971). In general, the endothelial cells lining the lumen of blood vessels are quiescent, and neovascularization is virtually absent. However, when these cells are stimulated they have early vascular neoformation, which can occur via two different mechanisms: vasculogenesis and angiogenesis (Lamalice et al., 2007; Adams and Alitalo, 2007). Angiogenesis is the formation of a new network of vessels from preexisting vessels, a process that occurs via budding or intussusception. For angiogenesis to occur, tissue stimulation is necessary to induce and increase the expression of pro-angiogenic factors such as Vascular Endothelial Factor (VEGF) and Fibroblast Growth Factor (Folkman and Klagsbrun, 1987; Dias et al., 2002). Angiogenesis is essential for the repair and development of tissues such as wound healing, the development of collateral circulation in ischemic tissues in corpus luteum, endometrium, placenta formation and hair growth. However, it can also be associated with pathological conditions such as cancer and eye changes (Adams and Alitalo, 2007; Folkman and Klagsbrun, 1987).
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The inflammatory process consists of a series of events that are related to tissue injury or infection. The inflammation, proliferation and remodeling of tissues are phases that occur in the synthesis and degradation of the extracellular matrix involving the activation and inhibition of the hyaluronidase enzyme (Bornstein and Sage, 2002). Hyaluronidase is an enzyme that hydrolyses hyaluronic acid, an acid whose function is to ensure that cells remain adhered to one other. Fragmentation of this polymer significantly decreases intracellular viscosity, facilitating the proliferation of these cells from the tissues, leading to a consequent degradation of the extracellular matrix that promotes inflammation (Jeong et al., 2000). Hyaluronidase can be inhibited by chemicals or by immunological methods using natural inhibitors such as phenolic compounds, flavonoids and esters, phenolic aldehydes, alcohols, etc. (Jeong et al., 2000; Salmen, 2003). This study aimed to evaluate the angiogenic and anti-inflammatory activity of polysaccharide extract obtained by fermentation of Tibetan kefir in whey. 2. Materials and methods 2.1. Obtaining the polysaccharide extract We obtained the bioactive compound to basic exopolysaccharide extract (ExPP) via fermentation of Tibetan kefir in a medium composed of whey supplemented with 30% glucose (w/v), 2% bacteriological peptone (w/v), 4% potassium phosphate monobasic (w/v) and 4% sodium citrate dehydrate (w/v); the fermentation medium was pasteurized at 63 °C for 30 min. All of the reagents that we used were analytical grade. The inoculum rate was 6% (w/v) of the 108 CFU/g consists of the following bacteria Lactobacillus kefiranofaciens, Lactobacillus helveticus, Lb. lactis, Lb. lactis subsp cremoris, Lb. lactis subsp lactis, Lb. casei, Lb. kefiri, Leuconostoc mesenteroides, Pseudomonas sp., Pseudomonas putida, Pseudomonas fluorecenses and 108 CFU/g consists of the following yeast Kazachstania unispora, Kazachstania exigua, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromices siamensis, Saccharomices cerevisae, Saccharomices unisporus, Saccharomices martiniae, Candida humilis, respectively, and fermentation time was conducted at 37 °C for 48 h. At the end of 48 h of fermentation, the fermented medium was centrifuged and filtered with 20% trichloroacetic (1:1) at 4000 g for 30 min at 4 °C. The supernatant was added to absolute ethanol (1:3) at 4 °C and stored at 4 °C for 24 h to precipitate the ExPP. Next, the centrifugation was repeated under the same conditions, as was the ethanol precipitation for an additional 24 h. After the second round of precipitation, the ExPP sample was centrifuged again under the same conditions, and we dialyzed the precipitate in a dialysis membrane of 10–12 kDa pore size (Sigma-Aldrich, St. Louis, MO, USA) against ultra-pure water for 72 h. Next, the dialyzed sample was frozen and lyophilized (freezedryer, Modulyo©, Thermo Electron, USA). 2.2. Monosaccharide composition of the extract polysaccharide Approximately 1 mg of lyophilized ExPP was subjected to total acid hydrolysis with 1 mL of 1 M trifluoroacetic acid for 4 h at 100 °C. Then, the lyophilized ExPP was reduced with sodium borohydride (NaBH4, 20 mg) for 12 h. After reduction, the content was neutralized (pH 5–6) with 50% v/v acetic acid and freeze-dried. The dried product was co-distilled with methanol to remove boron salts. Next, an acetylation step was performed with 1 mL acetic anhydride for 1 h at 120 °C, and we analyzed the monosaccharides as alditol acetate derivatives using gas chromatography-mass spectrometry (GC-MS) in a Varian 3800 chromatography system using helium as a mobile phase (1.0 mL/min) in a DB-225MS capillary column (Durabond, 30 m × 0.25 mm; internal diameter 0.25 μm). The peak areas were determined via integration using the Varian software MS Data Review. All of the reagents were analytical grade unless otherwise specified.
2.3. Evaluation of angiogenic activity 2.3.1. Animals We obtained fertilized eggs of Gallus gallus from a commercial poultry farm located in the Curitiba metropolitan region of Brazil. We maintained the eggs at ambient temperature (above 20 °C) until use. This project was approved by the Ethical Committee for Animal Experimentation of the Federal University of Paraná and registered under number 23,075.047773/2010-61. 2.3.2. Samples evaluated in biological models We evaluated the biological activity of ExPP according to the pharmacological model for the dose-response curve. The polysaccharides were used in the ExPP in lyophilized (L), frozen (F), dialysate (D) and precipitate (Pt) forms, as necessary. 2.3.3. Evaluation of angiogenic activity To evaluate the angiogenic activity of the polysaccharide extract, we used a modified chicken chorioallantoic membrane ex ovo assay (Boller et al., 2015). The study involved the evaluation of the ExPP (L) on a chorioallantoic membrane CAM capillary vessel in order to determine if the extract was angiogenic or antiangiogenic. In this assay, egg embryos were incubated at 37 °C and 60% relative humidity for 10 days. After that a 6 mm cellulose disc was laid over CAM with 10 μL of ExPP (L), VEGF (positive control) and hydrocortisone (HC – negative control) at 1, 3, 10, 30, 100, 300, 1000 and 3000 ng/mL. All analysis were made in triplicate and returned to the incubation chamber for a further 2 days. The egg embryos were removed from incubation and a photomicrography was taken of each disc to count the total number of blood vessels surrounding the disc. This process was accomplished using a 30% milk powder solution injected inside the CAM sack and submitted to image analysis using the Image J program. The total blood vessels for each disc were plotted graphically (Graph Pad Prism, version 5.0) to obtain either 50% inhibition (IC50) or excitatory (EC50) concentrations. 2.4. Evaluation of anti-inflammatory activity in vitro The polysaccharide (ExPP) was first tested in different presentation forms in order to review the best anti-inflammatory response by inhibiting the enzyme hyaluronidase. We studied the ExPP precipitated (ExPP (Pt)) with ethanol, ExPP lyophilized (ExPP (L)) and ExPP dialyzed (ExPP (D)) for 72 h against water and ExPP frozen (ExPP (F)) at the − 80 °C and were tested at a concentration of 5 mg/mL. DMSO was used as positive control due to its ability to completely inhibit de hyaluronidase enzyme. Although not used as anti-inflammatory agent, it can simulate an effective anti-inflammatory substance. We also used a natural anti-inflammatory agent as positive control, propolis commercial extract, at same concentration (Reissing et al., 1955; Arossos and Davidson, 1967; Kuppusamy et al., 1990). We determined the inhibition of hyaluronidase activity (Reissing et al., 1955; Arossos and Davidson, 1967; Kuppusamy et al., 1990). For the sample analysis, we placed 50 μL of ExPP (L) in different concentration (1, 2, 3, 5, 7 and 8 mg/mL) and 0.5 mL of potassium salt of hyaluronic acid (Sigma-Aldrich, St.Louis, MO, USA) (1.2 mg hyaluronic acid per mL of 0.1 M acetate buffer, pH 3.6, containing 0.15 M NaCl) in a reaction tube. The control tube consisted of the same reagent of test tubes without ExPP. All tube were incubated for 5 min at 37 °C and after that 50 μL of the enzyme hyaluronidase (350 units of the enzyme hyaluronidase type IV-S from bovine testes, Sigma-Aldrich, St.Louis, MO, USA - dissolved in the same buffer substrate at concentration 6.5 mg/mL) was added, and incubated at 37 °C for 40 min. The reaction was stopped by adding 10 L of 4 N sodium hydroxide solution and immediately placing 0.1 mL of 0.8 M potassium tetraborate into the reaction mixture and incubating it in a boiling bath for 3 min. After the incubation time, we added 3 mL of 4-dimethylaminobenzaldehyde
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(DMAB) (10% solution in glacial acetic acid containing 12.5% 10 N hydrochloric acid) to the reacted mixture and incubated the solution at 37 °C for 20 min. Next, we measured the samples in a spectrophotometer (SP 2000 UV Spectrum) at 585 nm. All of the reagents were analytical grade unless otherwise specified. 2.5. Cytotoxicity assay We performed cytotoxicity assays using kidney epithelial cells VERO from Cercopithecus aethiops donated by the Instituto de Tecnologia do Paraná (TECPAR). VERO cells were determined via 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, and we plated cells (1 × 105) in 0.2 mL of medium per well in 96-well plates, and incubated under 5% CO2 incubator during all stages. After 24 h, the medium was removed and serial dilution of ExPP L was added at 1, 3, 10, 30, 100, 300, 1000 and 3000 ng/mL (triplicate). This system was incubated for 24 h and after that a MTT assay was performed to verify cell viability. For the MTT assay, the medium from the wells was removed carefully after incubation. Each well was washed with minimum essential medium eagle (w/o) fetal bovine serum 2–3 times; we then added 200 μL of MTT (5 mg/mL). The plates were incubated for 6–7 h and after that, 1 mL of dimethylsulfoxide (DMSO) (solubilizing reagent) was added to each well and mixed completely using a micropipette and left for 45 s. We visualized the presence of viable cells by the development of a purple color due to the formation of formazan crystals. The suspension was transferred to the cuvette of a spectrophotometer and the optical density (OD) values were read at 595 nm using DMSO as a blank.
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monosaccharide (Badel et al., 2011). Heteropolysaccharides are made by polymerizing repeating unit precursors formed in the cytoplasm (Ahmed et al., 2013), requiring a medium rich in carbohydrates. We determined the monosaccharide composition of the ExPP (L) produced by Lactobacillus kefiranofaciens Zw3 isolated from Tibetan kefir; we identified only the presence of galactose and glucose heteropolysaccharide (Wang et al., 2008). This composition is similar to that found in previous studies of the type of ExPP (L) L. kefiranofaciens isolated from kefir (Maeda et al., 2004). 3.2. Evaluation of angiogenic activity
3. Results and discussion
The biological effect of a substance can be described as a function of its efficacy and potency; this effect can be determined by a doseresponse relationship of the modulating effect on a biological target. We obtained the concentration-effect curve of the different substances by counting the vessels intercepting the disk with the substances being tested. The ratio was adjusted by a sigmoid function to estimate the EC50 for ExPP (L) and VEGF and the inhibitory concentration (IC50) for HC. In terms of ExPP (L), we recovered an EC50 of 192 ng/mL (with a confidence level of 95%). For VEGF, the corresponding number was 5.562 ng/mL (Fig. 1) (with a confidence level of 95%). We can accordingly state that ExPP (L) can be considered to be a good pro-angiogenic agent because it augmented by 160% the formation of new vessels similar to VEGF (170%). To perform this effect, the concentration of ExPP (L) should be 28.9 higher than VEGF. After conducting statistical analyses (ANOVA) using the Bonferroni post-test, it was possible to observe a significant difference between the EC50 of VEGF and ExPP (with p b 0.05). Hydrocortisone is a steroid with anti-angiogenic activity. This substance was tested as a negative control of angiogenesis in order to evaluate the behavior of ExPP (L) with an IC50 of 1.222 mg/mL. Based on Fig. 2, one can see that ExPP (L) and HC exhibited very difficult behaviors regarding the percentage of the number of new vessels, confirming the anti-angiogenic behavior of HC and the pro-angiogenic behavior ExPP (L) in terms of modulating the activity of angiogenesis. These data allow us to infer that the ExPP (L) possesses angiogenic activity but not antiangiogenic activity.
3.1. Monosaccharide composition of the EPS
3.3. Evaluation of anti-inflammatory activity
We analyzed the ExPP (L) obtained from fermentation in Tibetan kefir using a medium supplemented with whey. Our goal was to determine the monosaccharide composition. As shown in Table 1, the ExPP (L) study revealed a higher concentration of galactose, glucose and mannose, and very small concentrations of rhamnose and arabinose. Based on the presence of different monosaccharides, it is likely that the ExPP (L) produced in this work is classifiable as a heteropolysaccharide. The heteropolysaccharides produced by lactic acid bacteria are mainly repeating galactose, glucose and rhamnose to the major monosaccharides, but they may also consist of other monosaccharides (Harding et al., 2005; Sánchez et al., 2006). They are often highly branched with different types of connections. Designations for the heteropolysaccharides are complex and depend on the primary
We analyzed our results using variance analysis (ANOVA) with 95% confidence (p b 0.05) and a post-test of Tukey to compare the samples with one other. Fig. 3 shows the anti-inflammatory activity of the different samples compared with DMSO. The ExPP exhibited a good anti-inflammatory response. When one compares ExPP (L) (63% activity) and ExPP (D) (61% activity), it is
2.6. Statistical analysis To statistically assess the results, we used a normality test, analysis of variance (ANOVA) and non-linear regression of the different ExPP concentrations on angiogenic tests to determinate the stimulatory and inhibitory concentration of the exopolysaccharide (EC50 and IC50). We also then used a post-test in the program GraphPad Prism 5.0 (GraphPad Software, Inc.; La Jolla, CA, USA).
Table 1 Monosaccharide composition of the ExPP (L) produced by Tibetan kefir. Monosaccharide
Percentage (%)a
Galactose Glucose Mannose Arabinose Rhamnose
39.00 ± 0.56 28.00 ± 0.65 26.00 ± 0.25 4.00 ± 0.21 3.00 ± 0.10
a
The values are means ± standard deviations (n = 3).
Fig. 1. Curve-response for VEGF and ExPP L. Vessel formation of CAM was augmented by VEGF and ExPP (L) with an EC50 of 5.562 ng/mL and 192 ng/mL, respectively with p b 0,05.
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Fig. 2. Curve-response for hydrocortisone and ExPP L. Vessel formation of CAM was augmented ExPP (L) and reduced by hydrocortisone with an EC50 of 192 ng/mL and IC50 of 1.222 mg/mL, respectively with p b 0,05.
obvious that there is no significant difference (p b 0.05) between them. However, ExPP (L) is more stable because it contains no water. When we analyzed ExPP (Pt) (45% activity) and ExPP (C) (45% activity) compared with ExPP (L), the former two materials exhibited highly significant differences at the level of 95% confidence (p b 0.05). Therefore, ExPP (Pt) and ExPP (C) are considered to be poor anti-inflammatory substances. ExPP (L) was chosen as the best presentation form by its relative anti-inflammatory activity by inhibiting the enzyme hyaluronidase, and this extract was tested at concentrations of 1, 2, 3, 5, 7 and 8 mg/mL to define the best inhibitory concentration of hyaluronidase enzyme and therefore the inflammatory process. Our results are shown in Fig. 4 in terms of log concentration to facilitate identification of the concentration defined as the inhibitory dose response (IC50), the concentration that induces 50% of the maximum inhibition of the enzyme hyaluronidase with an IC50 of 2.31 mg/mL. The antiinflammatory activity of Tibetan kefir in the rat paw edema method revealed a 43% decrease in the inflammatory process, suggesting a therapeutic potential for the kefir suspension as an anti-inflammatory agent. To provide more conclusive results regarding the inhibitory capacity of hyaluronidase in the inflammatory process, we compared ExPP
Fig. 3. Evaluation of anti-inflammatory activity of the different ExPP. ExPP showed an antiinflammatory activity in all its form (D, L, Pt and C) with p b 0,05. The statistical difference between the different extract are shown in letters, were same letter show no statistical difference and different letter have p b 0,05. DMSO values are statistical different from all other groups and is represented by *.
Fig. 4. Inhibitory dose response curve of ExPP (L). The graphic shows an inhibition of the hyaluronidase enzyme by ExPP (L) with an IC50 of 2.31 mg/mL.
(L) with a natural commercial product composed of the ethanolic extract of propolis. For this evaluation, we tested the ExPP (L) and the ethanol extract of the commercial propolis at the same concentrations. The results were analyzed statistically using ANOVA followed by a post-test of Tukey, and we compared the inhibitory activity of the hyaluronidase ExPP (L) with the inhibitory activity of hyaluronidase of the ethanol extract of propolis. Significant differences were observed (p b 0.05), and the ExPP (L) exhibited a maximum activity of 67.1% at a concentration of 7 mg/mL; the ethanolic extract of propolis exhibited a maximum activity of 58.0% at a concentration of 8 mg/mL, as shown in Fig. 5. These results suggest that kefir polysaccharides have a higher anti-inflammatory activity with lower dosages than propolis ethanolic extract.
3.4. Cytotoxicity assessment Cytotoxicity is the set of changes of cellular homeostasis that lead to a number of modifications that interfere with the adaptive ability of cells as well as their survival, reproduction, and carrying out of their metabolic functions (Todryk et al., 2001). A cytotoxicity test is an important way to determine the toxicity and safety of a substance that is already being investigated for its biological activity, and these tests are conducted in the early stages of developing a new product that has therapeutic potential (Sánchez et al., 2006; Badel et al., 2011).
Fig. 5. Comparison between the anti-inflammatory activity of ExPP (L) and propolis ethanolic extract. Both samples (ExPP (L) and propolis have anti-inflammatory activity. The statistical difference between the different samples are shown in letters, were same letter show no statistical difference and different letter have p b 0,05.
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Fig. 6. Cytotoxicity test for ExPP (L). The graphic show no significative death of VERO cells on MTT assay, meaning a non-toxic effect of ExPP (L) on VERO cells.
In vitro cytotoxicity tests, which are the most commonly used, evaluate basal cytotoxicity, enabling a definition of the limit of toxic concentration. In general, the method is based on cell permeability change using dyes (Sánchez et al., 2006; Ahmed et al., 2013). The MTT, when incubated with living cells, has its substrate broken via mitochondrial enzymes such as dehydrogenases, and it changes from a yellow compound to a dark blue compound (formazan). The production of formazan reflects the functional status of the respiratory chain and cell viability. The ability of cells to reduce the MTT provides an indication of the activity and mitochondrial integrity, which are interpreted as cell viability measures (Wang et al., 2008; Maeda et al., 2004). We analyzed our data to obtain the concentration-effect cytotoxicity in terms of the number of percentage of viable cells measured by the spectrophotometric method. The test revealed a non-significant reduction in cell growth (Fig. 6). As a result, ExPP (L) is considered to be safe in terms of cytotoxicity. 4. Conclusion ExPP (L) can be classified as a pro-angiogenic agent in modulating the angiogenesis process; it exhibits a behavior similar to that of VEGF. Regarding the hyaluronidase inhibitory activity of the compound in the inflammation process, the lyophilized polysaccharide exhibited good inhibitory action of hyaluronidase. Compared with a commercial product, it demonstrated superior action of ExPP (L) wich allows it to be used as pro-angiogenic and anti-inflammatory compound. Furthermore, this lyophilized polysaccharide extract is a safe compound with respect to cytotoxicity, enabling its use in different products. Polysaccharides are macromolecules that are not suitable for oral or endovenous medicine. Therefore, a possible pharmaceutical product using the polysaccharides isolated from Tibetan kefir should be design for topical use, i.e. in form of gel, balm, cream. In this aspect, future work should include pharmaceutical formulation and stability tests. References Adams, R.H., Alitalo, K., 2007. Molecular regulation of angiogenesis and lymphangiogenesis. Nature Reviews 8, 464–478. Ahmed, Z., Wang, Y., Anjum, N., Ahmadc, A., Khan, S.T., 2013. Characterization of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir - part II. Food Hydrocoll. 2013 (30), 343–350.
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Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
Anti-inflammatory and angiogenic activity of polysaccharide extract obtained from Tibetan kefir Maria Rosa Machado Prado a,⁎, Christian Boller a, Rosiane Guetter Mello Zibetti a,c, Daiany de Souza c, Luciana Lopes Pedroso b, Carlos Ricardo Soccol d a
Faculdades Pequeno Príncipe, Curitiba, PR, Brazil Instituto de Tecnologia do Paraná, Paraná, Curitiba, PR, Brazil Instituto de Pesquisa Pelé Pequeno Príncipe de Pesquisa, Curitiba, PR, Brazil d Universidade Federal do Paraná, Curitiba, PR, Brazil b c
a r t i c l e
i n f o
Article history: Received 19 April 2016 Revised 4 July 2016 Accepted 10 July 2016 Available online 11 July 2016 Keywords: Tibetan kefir Angiogenic activity Anti-inflammatory activity Hyaluronidase
a b s t r a c t The search for new bioactive molecules is a driving force for research pharmaceutical industries, especially those molecules obtained from fermentation. The molecules possessing angiogenic and anti-inflammatory attributes have attracted attention and are the focus of this study. Angiogenic activity from kefir polysaccharide extract, via chorioallantoic membrane assay, exhibited a pro-angiogenic effect compared with vascular endothelial factor (pro-angiogenic) and hydrocortisone (anti-angiogenic) activity as standards with an EC50 of 192 ng/mL. In terms of anti-inflammatory activity determined via hyaluronidase enzyme assay, kefir polysaccharide extract inhibited the enzyme with a minimal activity of 2.08 mg/mL and a maximum activity of 2.57 mg/mL. For pharmaceutical purposes, kefir polysaccharide extract is considered to be safe because it does not inhibit VERO cells in cytotoxicity assays. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Many studies have been conducted with different molecules, extracts and products obtained by fermentation in order to reveal and confirm their biological activity and use in the development of new pharmaceutical, cosmetic and nutraceutical products. Kefir is a fermented milk product with an acidic taste and a creamy consistency that originated in the Balkans, Eastern Europe and the Caucasus (Fontán et al., 2006; Serafini et al., 2014). Kefir has beneficial effects on human health thanks to its antimicrobial (Anselmo et al., 2010), immunoregulatory (Hong et al., 2009), antiallergenic (WeiSheng et al., 2010), antitumoral (Gao et al., 2013), anti-inflammatory (Diniz et al., 2003), antidiabetic (Young-In et al., 2006) and antimutagenic (Guzel-Seydim et al., 2006) activities. The bioactivities of the kefir are attributed to the microorganisms and the substances within it, such as exopolysaccharides produced during the fermentation process of kefir grains. Polysaccharides obtained from different sources, such as plants, animals and microorganisms, have been studied for possessing different biological activities: antibiotic, antioxidant, anticoagulant, anti-mutant,
⁎ Corresponding author at: Faculdades Pequeno Príncipe, Avenida Iguaçu 333, Curitiba, PR 80230-020, Brazil. E-mail address: [email protected] (M.R.M. Prado).
http://dx.doi.org/10.1016/j.mvr.2016.07.004 0026-2862/© 2016 Elsevier Inc. All rights reserved.
immuno-stimulation and anti-cancer (Yu et al., 2009; Wijesekara et al., 2011; Zong et al., 2012). However, polysaccharides derived from microorganisms—especially bacteria—are still being assessed for these activities and others (e.g., angiogenesis and anti-inflammatory). The first studies on angiogenesis were proposed by Folkman (1971) who applied the concept of vascular bed control (i.e., stimulation or inhibition of new vessels) in an attempt to elucidate this process and also as an alternative treatment for various medical conditions (Folkman, 1971). In general, the endothelial cells lining the lumen of blood vessels are quiescent, and neovascularization is virtually absent. However, when these cells are stimulated they have early vascular neoformation, which can occur via two different mechanisms: vasculogenesis and angiogenesis (Lamalice et al., 2007; Adams and Alitalo, 2007). Angiogenesis is the formation of a new network of vessels from preexisting vessels, a process that occurs via budding or intussusception. For angiogenesis to occur, tissue stimulation is necessary to induce and increase the expression of pro-angiogenic factors such as Vascular Endothelial Factor (VEGF) and Fibroblast Growth Factor (Folkman and Klagsbrun, 1987; Dias et al., 2002). Angiogenesis is essential for the repair and development of tissues such as wound healing, the development of collateral circulation in ischemic tissues in corpus luteum, endometrium, placenta formation and hair growth. However, it can also be associated with pathological conditions such as cancer and eye changes (Adams and Alitalo, 2007; Folkman and Klagsbrun, 1987).
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The inflammatory process consists of a series of events that are related to tissue injury or infection. The inflammation, proliferation and remodeling of tissues are phases that occur in the synthesis and degradation of the extracellular matrix involving the activation and inhibition of the hyaluronidase enzyme (Bornstein and Sage, 2002). Hyaluronidase is an enzyme that hydrolyses hyaluronic acid, an acid whose function is to ensure that cells remain adhered to one other. Fragmentation of this polymer significantly decreases intracellular viscosity, facilitating the proliferation of these cells from the tissues, leading to a consequent degradation of the extracellular matrix that promotes inflammation (Jeong et al., 2000). Hyaluronidase can be inhibited by chemicals or by immunological methods using natural inhibitors such as phenolic compounds, flavonoids and esters, phenolic aldehydes, alcohols, etc. (Jeong et al., 2000; Salmen, 2003). This study aimed to evaluate the angiogenic and anti-inflammatory activity of polysaccharide extract obtained by fermentation of Tibetan kefir in whey. 2. Materials and methods 2.1. Obtaining the polysaccharide extract We obtained the bioactive compound to basic exopolysaccharide extract (ExPP) via fermentation of Tibetan kefir in a medium composed of whey supplemented with 30% glucose (w/v), 2% bacteriological peptone (w/v), 4% potassium phosphate monobasic (w/v) and 4% sodium citrate dehydrate (w/v); the fermentation medium was pasteurized at 63 °C for 30 min. All of the reagents that we used were analytical grade. The inoculum rate was 6% (w/v) of the 108 CFU/g consists of the following bacteria Lactobacillus kefiranofaciens, Lactobacillus helveticus, Lb. lactis, Lb. lactis subsp cremoris, Lb. lactis subsp lactis, Lb. casei, Lb. kefiri, Leuconostoc mesenteroides, Pseudomonas sp., Pseudomonas putida, Pseudomonas fluorecenses and 108 CFU/g consists of the following yeast Kazachstania unispora, Kazachstania exigua, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromices siamensis, Saccharomices cerevisae, Saccharomices unisporus, Saccharomices martiniae, Candida humilis, respectively, and fermentation time was conducted at 37 °C for 48 h. At the end of 48 h of fermentation, the fermented medium was centrifuged and filtered with 20% trichloroacetic (1:1) at 4000 g for 30 min at 4 °C. The supernatant was added to absolute ethanol (1:3) at 4 °C and stored at 4 °C for 24 h to precipitate the ExPP. Next, the centrifugation was repeated under the same conditions, as was the ethanol precipitation for an additional 24 h. After the second round of precipitation, the ExPP sample was centrifuged again under the same conditions, and we dialyzed the precipitate in a dialysis membrane of 10–12 kDa pore size (Sigma-Aldrich, St. Louis, MO, USA) against ultra-pure water for 72 h. Next, the dialyzed sample was frozen and lyophilized (freezedryer, Modulyo©, Thermo Electron, USA). 2.2. Monosaccharide composition of the extract polysaccharide Approximately 1 mg of lyophilized ExPP was subjected to total acid hydrolysis with 1 mL of 1 M trifluoroacetic acid for 4 h at 100 °C. Then, the lyophilized ExPP was reduced with sodium borohydride (NaBH4, 20 mg) for 12 h. After reduction, the content was neutralized (pH 5–6) with 50% v/v acetic acid and freeze-dried. The dried product was co-distilled with methanol to remove boron salts. Next, an acetylation step was performed with 1 mL acetic anhydride for 1 h at 120 °C, and we analyzed the monosaccharides as alditol acetate derivatives using gas chromatography-mass spectrometry (GC-MS) in a Varian 3800 chromatography system using helium as a mobile phase (1.0 mL/min) in a DB-225MS capillary column (Durabond, 30 m × 0.25 mm; internal diameter 0.25 μm). The peak areas were determined via integration using the Varian software MS Data Review. All of the reagents were analytical grade unless otherwise specified.
2.3. Evaluation of angiogenic activity 2.3.1. Animals We obtained fertilized eggs of Gallus gallus from a commercial poultry farm located in the Curitiba metropolitan region of Brazil. We maintained the eggs at ambient temperature (above 20 °C) until use. This project was approved by the Ethical Committee for Animal Experimentation of the Federal University of Paraná and registered under number 23,075.047773/2010-61. 2.3.2. Samples evaluated in biological models We evaluated the biological activity of ExPP according to the pharmacological model for the dose-response curve. The polysaccharides were used in the ExPP in lyophilized (L), frozen (F), dialysate (D) and precipitate (Pt) forms, as necessary. 2.3.3. Evaluation of angiogenic activity To evaluate the angiogenic activity of the polysaccharide extract, we used a modified chicken chorioallantoic membrane ex ovo assay (Boller et al., 2015). The study involved the evaluation of the ExPP (L) on a chorioallantoic membrane CAM capillary vessel in order to determine if the extract was angiogenic or antiangiogenic. In this assay, egg embryos were incubated at 37 °C and 60% relative humidity for 10 days. After that a 6 mm cellulose disc was laid over CAM with 10 μL of ExPP (L), VEGF (positive control) and hydrocortisone (HC – negative control) at 1, 3, 10, 30, 100, 300, 1000 and 3000 ng/mL. All analysis were made in triplicate and returned to the incubation chamber for a further 2 days. The egg embryos were removed from incubation and a photomicrography was taken of each disc to count the total number of blood vessels surrounding the disc. This process was accomplished using a 30% milk powder solution injected inside the CAM sack and submitted to image analysis using the Image J program. The total blood vessels for each disc were plotted graphically (Graph Pad Prism, version 5.0) to obtain either 50% inhibition (IC50) or excitatory (EC50) concentrations. 2.4. Evaluation of anti-inflammatory activity in vitro The polysaccharide (ExPP) was first tested in different presentation forms in order to review the best anti-inflammatory response by inhibiting the enzyme hyaluronidase. We studied the ExPP precipitated (ExPP (Pt)) with ethanol, ExPP lyophilized (ExPP (L)) and ExPP dialyzed (ExPP (D)) for 72 h against water and ExPP frozen (ExPP (F)) at the − 80 °C and were tested at a concentration of 5 mg/mL. DMSO was used as positive control due to its ability to completely inhibit de hyaluronidase enzyme. Although not used as anti-inflammatory agent, it can simulate an effective anti-inflammatory substance. We also used a natural anti-inflammatory agent as positive control, propolis commercial extract, at same concentration (Reissing et al., 1955; Arossos and Davidson, 1967; Kuppusamy et al., 1990). We determined the inhibition of hyaluronidase activity (Reissing et al., 1955; Arossos and Davidson, 1967; Kuppusamy et al., 1990). For the sample analysis, we placed 50 μL of ExPP (L) in different concentration (1, 2, 3, 5, 7 and 8 mg/mL) and 0.5 mL of potassium salt of hyaluronic acid (Sigma-Aldrich, St.Louis, MO, USA) (1.2 mg hyaluronic acid per mL of 0.1 M acetate buffer, pH 3.6, containing 0.15 M NaCl) in a reaction tube. The control tube consisted of the same reagent of test tubes without ExPP. All tube were incubated for 5 min at 37 °C and after that 50 μL of the enzyme hyaluronidase (350 units of the enzyme hyaluronidase type IV-S from bovine testes, Sigma-Aldrich, St.Louis, MO, USA - dissolved in the same buffer substrate at concentration 6.5 mg/mL) was added, and incubated at 37 °C for 40 min. The reaction was stopped by adding 10 L of 4 N sodium hydroxide solution and immediately placing 0.1 mL of 0.8 M potassium tetraborate into the reaction mixture and incubating it in a boiling bath for 3 min. After the incubation time, we added 3 mL of 4-dimethylaminobenzaldehyde
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(DMAB) (10% solution in glacial acetic acid containing 12.5% 10 N hydrochloric acid) to the reacted mixture and incubated the solution at 37 °C for 20 min. Next, we measured the samples in a spectrophotometer (SP 2000 UV Spectrum) at 585 nm. All of the reagents were analytical grade unless otherwise specified. 2.5. Cytotoxicity assay We performed cytotoxicity assays using kidney epithelial cells VERO from Cercopithecus aethiops donated by the Instituto de Tecnologia do Paraná (TECPAR). VERO cells were determined via 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, and we plated cells (1 × 105) in 0.2 mL of medium per well in 96-well plates, and incubated under 5% CO2 incubator during all stages. After 24 h, the medium was removed and serial dilution of ExPP L was added at 1, 3, 10, 30, 100, 300, 1000 and 3000 ng/mL (triplicate). This system was incubated for 24 h and after that a MTT assay was performed to verify cell viability. For the MTT assay, the medium from the wells was removed carefully after incubation. Each well was washed with minimum essential medium eagle (w/o) fetal bovine serum 2–3 times; we then added 200 μL of MTT (5 mg/mL). The plates were incubated for 6–7 h and after that, 1 mL of dimethylsulfoxide (DMSO) (solubilizing reagent) was added to each well and mixed completely using a micropipette and left for 45 s. We visualized the presence of viable cells by the development of a purple color due to the formation of formazan crystals. The suspension was transferred to the cuvette of a spectrophotometer and the optical density (OD) values were read at 595 nm using DMSO as a blank.
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monosaccharide (Badel et al., 2011). Heteropolysaccharides are made by polymerizing repeating unit precursors formed in the cytoplasm (Ahmed et al., 2013), requiring a medium rich in carbohydrates. We determined the monosaccharide composition of the ExPP (L) produced by Lactobacillus kefiranofaciens Zw3 isolated from Tibetan kefir; we identified only the presence of galactose and glucose heteropolysaccharide (Wang et al., 2008). This composition is similar to that found in previous studies of the type of ExPP (L) L. kefiranofaciens isolated from kefir (Maeda et al., 2004). 3.2. Evaluation of angiogenic activity
3. Results and discussion
The biological effect of a substance can be described as a function of its efficacy and potency; this effect can be determined by a doseresponse relationship of the modulating effect on a biological target. We obtained the concentration-effect curve of the different substances by counting the vessels intercepting the disk with the substances being tested. The ratio was adjusted by a sigmoid function to estimate the EC50 for ExPP (L) and VEGF and the inhibitory concentration (IC50) for HC. In terms of ExPP (L), we recovered an EC50 of 192 ng/mL (with a confidence level of 95%). For VEGF, the corresponding number was 5.562 ng/mL (Fig. 1) (with a confidence level of 95%). We can accordingly state that ExPP (L) can be considered to be a good pro-angiogenic agent because it augmented by 160% the formation of new vessels similar to VEGF (170%). To perform this effect, the concentration of ExPP (L) should be 28.9 higher than VEGF. After conducting statistical analyses (ANOVA) using the Bonferroni post-test, it was possible to observe a significant difference between the EC50 of VEGF and ExPP (with p b 0.05). Hydrocortisone is a steroid with anti-angiogenic activity. This substance was tested as a negative control of angiogenesis in order to evaluate the behavior of ExPP (L) with an IC50 of 1.222 mg/mL. Based on Fig. 2, one can see that ExPP (L) and HC exhibited very difficult behaviors regarding the percentage of the number of new vessels, confirming the anti-angiogenic behavior of HC and the pro-angiogenic behavior ExPP (L) in terms of modulating the activity of angiogenesis. These data allow us to infer that the ExPP (L) possesses angiogenic activity but not antiangiogenic activity.
3.1. Monosaccharide composition of the EPS
3.3. Evaluation of anti-inflammatory activity
We analyzed the ExPP (L) obtained from fermentation in Tibetan kefir using a medium supplemented with whey. Our goal was to determine the monosaccharide composition. As shown in Table 1, the ExPP (L) study revealed a higher concentration of galactose, glucose and mannose, and very small concentrations of rhamnose and arabinose. Based on the presence of different monosaccharides, it is likely that the ExPP (L) produced in this work is classifiable as a heteropolysaccharide. The heteropolysaccharides produced by lactic acid bacteria are mainly repeating galactose, glucose and rhamnose to the major monosaccharides, but they may also consist of other monosaccharides (Harding et al., 2005; Sánchez et al., 2006). They are often highly branched with different types of connections. Designations for the heteropolysaccharides are complex and depend on the primary
We analyzed our results using variance analysis (ANOVA) with 95% confidence (p b 0.05) and a post-test of Tukey to compare the samples with one other. Fig. 3 shows the anti-inflammatory activity of the different samples compared with DMSO. The ExPP exhibited a good anti-inflammatory response. When one compares ExPP (L) (63% activity) and ExPP (D) (61% activity), it is
2.6. Statistical analysis To statistically assess the results, we used a normality test, analysis of variance (ANOVA) and non-linear regression of the different ExPP concentrations on angiogenic tests to determinate the stimulatory and inhibitory concentration of the exopolysaccharide (EC50 and IC50). We also then used a post-test in the program GraphPad Prism 5.0 (GraphPad Software, Inc.; La Jolla, CA, USA).
Table 1 Monosaccharide composition of the ExPP (L) produced by Tibetan kefir. Monosaccharide
Percentage (%)a
Galactose Glucose Mannose Arabinose Rhamnose
39.00 ± 0.56 28.00 ± 0.65 26.00 ± 0.25 4.00 ± 0.21 3.00 ± 0.10
a
The values are means ± standard deviations (n = 3).
Fig. 1. Curve-response for VEGF and ExPP L. Vessel formation of CAM was augmented by VEGF and ExPP (L) with an EC50 of 5.562 ng/mL and 192 ng/mL, respectively with p b 0,05.
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Fig. 2. Curve-response for hydrocortisone and ExPP L. Vessel formation of CAM was augmented ExPP (L) and reduced by hydrocortisone with an EC50 of 192 ng/mL and IC50 of 1.222 mg/mL, respectively with p b 0,05.
obvious that there is no significant difference (p b 0.05) between them. However, ExPP (L) is more stable because it contains no water. When we analyzed ExPP (Pt) (45% activity) and ExPP (C) (45% activity) compared with ExPP (L), the former two materials exhibited highly significant differences at the level of 95% confidence (p b 0.05). Therefore, ExPP (Pt) and ExPP (C) are considered to be poor anti-inflammatory substances. ExPP (L) was chosen as the best presentation form by its relative anti-inflammatory activity by inhibiting the enzyme hyaluronidase, and this extract was tested at concentrations of 1, 2, 3, 5, 7 and 8 mg/mL to define the best inhibitory concentration of hyaluronidase enzyme and therefore the inflammatory process. Our results are shown in Fig. 4 in terms of log concentration to facilitate identification of the concentration defined as the inhibitory dose response (IC50), the concentration that induces 50% of the maximum inhibition of the enzyme hyaluronidase with an IC50 of 2.31 mg/mL. The antiinflammatory activity of Tibetan kefir in the rat paw edema method revealed a 43% decrease in the inflammatory process, suggesting a therapeutic potential for the kefir suspension as an anti-inflammatory agent. To provide more conclusive results regarding the inhibitory capacity of hyaluronidase in the inflammatory process, we compared ExPP
Fig. 3. Evaluation of anti-inflammatory activity of the different ExPP. ExPP showed an antiinflammatory activity in all its form (D, L, Pt and C) with p b 0,05. The statistical difference between the different extract are shown in letters, were same letter show no statistical difference and different letter have p b 0,05. DMSO values are statistical different from all other groups and is represented by *.
Fig. 4. Inhibitory dose response curve of ExPP (L). The graphic shows an inhibition of the hyaluronidase enzyme by ExPP (L) with an IC50 of 2.31 mg/mL.
(L) with a natural commercial product composed of the ethanolic extract of propolis. For this evaluation, we tested the ExPP (L) and the ethanol extract of the commercial propolis at the same concentrations. The results were analyzed statistically using ANOVA followed by a post-test of Tukey, and we compared the inhibitory activity of the hyaluronidase ExPP (L) with the inhibitory activity of hyaluronidase of the ethanol extract of propolis. Significant differences were observed (p b 0.05), and the ExPP (L) exhibited a maximum activity of 67.1% at a concentration of 7 mg/mL; the ethanolic extract of propolis exhibited a maximum activity of 58.0% at a concentration of 8 mg/mL, as shown in Fig. 5. These results suggest that kefir polysaccharides have a higher anti-inflammatory activity with lower dosages than propolis ethanolic extract.
3.4. Cytotoxicity assessment Cytotoxicity is the set of changes of cellular homeostasis that lead to a number of modifications that interfere with the adaptive ability of cells as well as their survival, reproduction, and carrying out of their metabolic functions (Todryk et al., 2001). A cytotoxicity test is an important way to determine the toxicity and safety of a substance that is already being investigated for its biological activity, and these tests are conducted in the early stages of developing a new product that has therapeutic potential (Sánchez et al., 2006; Badel et al., 2011).
Fig. 5. Comparison between the anti-inflammatory activity of ExPP (L) and propolis ethanolic extract. Both samples (ExPP (L) and propolis have anti-inflammatory activity. The statistical difference between the different samples are shown in letters, were same letter show no statistical difference and different letter have p b 0,05.
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Fig. 6. Cytotoxicity test for ExPP (L). The graphic show no significative death of VERO cells on MTT assay, meaning a non-toxic effect of ExPP (L) on VERO cells.
In vitro cytotoxicity tests, which are the most commonly used, evaluate basal cytotoxicity, enabling a definition of the limit of toxic concentration. In general, the method is based on cell permeability change using dyes (Sánchez et al., 2006; Ahmed et al., 2013). The MTT, when incubated with living cells, has its substrate broken via mitochondrial enzymes such as dehydrogenases, and it changes from a yellow compound to a dark blue compound (formazan). The production of formazan reflects the functional status of the respiratory chain and cell viability. The ability of cells to reduce the MTT provides an indication of the activity and mitochondrial integrity, which are interpreted as cell viability measures (Wang et al., 2008; Maeda et al., 2004). We analyzed our data to obtain the concentration-effect cytotoxicity in terms of the number of percentage of viable cells measured by the spectrophotometric method. The test revealed a non-significant reduction in cell growth (Fig. 6). As a result, ExPP (L) is considered to be safe in terms of cytotoxicity. 4. Conclusion ExPP (L) can be classified as a pro-angiogenic agent in modulating the angiogenesis process; it exhibits a behavior similar to that of VEGF. Regarding the hyaluronidase inhibitory activity of the compound in the inflammation process, the lyophilized polysaccharide exhibited good inhibitory action of hyaluronidase. Compared with a commercial product, it demonstrated superior action of ExPP (L) wich allows it to be used as pro-angiogenic and anti-inflammatory compound. Furthermore, this lyophilized polysaccharide extract is a safe compound with respect to cytotoxicity, enabling its use in different products. Polysaccharides are macromolecules that are not suitable for oral or endovenous medicine. Therefore, a possible pharmaceutical product using the polysaccharides isolated from Tibetan kefir should be design for topical use, i.e. in form of gel, balm, cream. In this aspect, future work should include pharmaceutical formulation and stability tests. References Adams, R.H., Alitalo, K., 2007. Molecular regulation of angiogenesis and lymphangiogenesis. Nature Reviews 8, 464–478. Ahmed, Z., Wang, Y., Anjum, N., Ahmadc, A., Khan, S.T., 2013. Characterization of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir - part II. Food Hydrocoll. 2013 (30), 343–350.
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