http://informahealthcare.com/enz ISSN: 1475-6366 (print), 1475-6374 (electronic) J Enzyme Inhib Med Chem, Early Online: 1–4 ! 2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1054819
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RESEARCH ARTICLE
Honey shows potent inhibitory activity against the bovine testes hyaluronidase Sevgi Kolayli1, Huseyin Sahin2, Zehra Can3, Oktay Yildiz4, and Ku¨bra Sahin1 1
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey, 2Espiye Vocational School, Giresun University, Espiye, Giresun, Turkey, 3¸Sebinkarahisar Technical Sciences Vocational School, Giresun University, Giresun, Turkey, and 4Mac¸ka Vocational School, Karadeniz Technical University, Mac¸ka, Trabzon, Turkey Abstract
Keywords
The purpose of this study was to investigate the anti-hyaluronidase activities of honeys from different botanical origins honeys in order to determine their anti-inflammatory properties. The total phenolic contents, total flavonoids and total tannin levels of six types of honey, chestnut, oak, heather, pine, buckwheat and mixed blossom, were determined. Concentrationrelated inhibition values were tested turbidimetrically on bovine testis hyaluronidase (BTHase) as IC50 (mg/mL). All honeys exhibited various concentration-dependent degrees of inhibition against BTHase. Inhibition values varied significantly depending on honeys’ levels of phenolic contents, flavonoid and tannin. The honeys with the highest anti-hyaluronidase activity were oak, chestnut and heather. In conclusion, polyphenol-rich honeys have high anti-hyaluronidase activity, and these honeys have high protective and complementary potential against hyaluronidase-induced anti-inflammatory failures.
Flavonoid, hyaluronidase, inhibition, phenolic content, tannin
Introduction Hyaluronidases are a class of enzymes which predominantly catalyze degradation of hyaluronic acid (HA) and are widespread in humans, bacteria and invertebrates1,2. HA or hyaluronan is a high molecular weight polysaccharide, the acidic glycosaminoglycan being composed of repeating disaccharide units of a-1,3 linked D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units2, does not contain sulfate groups, as well as heparin or chondroitin. The biopolymer is the main component of the extracellular matrix and connective tissues3,4. Concentration depends on the balance between HA syntheses via hyaluronate synthases and degradation via human hyaluronidases, mainly hyaluronidase 1 (hHyal-1), hyaluronidase 2 (hHyal-2) and PH-20 (hPH-20)5,6. The highly hydrophilic structure of HA means that it has many physiological roles, such as cell adhesion, migration and proliferation. Many studies have demonstrated the participation of HA in different physiological and pathophysiological conditions including embryogenesis, angiogenesis, wound healing, tissue turnover, malignancies and inflammatory disorders3,4. Hyaluronidases are involved in many pathological diseases, and their inhibitors serve as potential regulators as anti-inflammatory, anti-allergic, anti-tumoral, anti-aging, anti-rheumatoid, anti-toxin and antimicrobial agents4,7,8. The polysaccharide HA is important for tissue integrity, and has been reported to be associated with several diseases. HA fragments are reported to be Address for correspondence: Sevgi Kolayli, PhD, Department of Chemistry, Faculty of Science, Karadeniz Technical University, 61080 Trabzon, Turkey. Tel: +90 4623772487. Fax: +90 462325 3196. E-mail: [email protected]
History Received 21 March 2015 Revised 16 April 2015 Accepted 17 April 2015 Published online 15 June 2015
associated with cancer, and even to stimulate and facilitate cancer cell growth7,8. HA inhibitors have also led to new therapeutic concepts in pathophysiological conditions, such as laryngeal or breast cancer treatment, that are associated with the hyaluronan– hyaluronidase system3,4,7. Since the partial control of hyaluronidases possesses several important physiological functions, they have recently attracted considerable scientific interest as synthetic and natural inhibitors4,8–12. Honey and bee products are natural materials whose antioxidant, anti-inflammatory and anti-tumoral efficacies vary depending on the flora from which they are produced. Indeed, honey has been reported to have an inhibitory effect on monoamine oxidase13 and carbonic anhydrase14,15, and this is reported to derive more from honey polyphenols. No previous studies have investigated the anti-hyaluronidase activity of honey. This study for the first time investigated the anti-hyaluronidase activities of honey from various floral sources. The inhibition values obtained revealed a statistically significant association with honey’s phenolic contents.
Material and methods Reagents Bovine testes hyaluronidase (BTHase) (400–1000 U/mg solid), HAs odium salt and bovine serum albumin, sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic monohydrate (NaH2PO4H2O), gallic acid, sodium carbonate (Na2CO3), sodium acetate trihydrate (NaCH3COO3H2O) were purchased from Sigma-Aldrich (St. Louis, MO). Folin–Ciocalteu reagent was obtained from LiChrosolvÕ (Merck KGaA, Darmstadt, Germany).
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Table 1. Identification markers of Turkish honeys and concentration of phenolic substances and anti-hyaluronidase activities (HYA).
Code
Honey samples
Predominant pollen (%)
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17
Chestnut Chestnut Chestnut Heather Heather Heather Oak Oak Oak Pine Pine Pine Buckwheat Buckwheat Heterofloral Heterofloral Heterofloral
76 71 68 92 83 80 nd nd nd nd nd nd 62 65 nd nd nd
Familia Castanea sativa Mill. Calluna vulgaris Quercus L. Pinusburia L. Fagopyrume sculentum – – –
Total phenolic (mg GAE/100 g)
Total flavonoids (mg QUE/100 g)
Total tannin (mg tannic acid/100 g)
HYA (IC50 g/mL)
92.28 ± 2.25k 83.10 ± 3.13j 67.56 ± 2.74h 101.67 ± 3.51ij 92.00 ± 3.00k 90.67 ± 2.08i 80.33 ± 1.52f 91.00 ± 1.00g 78.00 ± 2.65d 41.33 ± 0.57e 48.33 ± 2.52c 36.67 ± 2.51l 56.33 ± 1.52k 60.32 ± 1.53k 26.23 ± 0.75b 27.66 ± 0.67b 19.07 ± 0.68a
7.60 ± 0.10l 6.50 ± 0.15k 6.0 ± 0.10j 1.32 ± 0.08h 1.18 ± 0.07i 1.13 ± 0.06g 4.80 ± 0.20d 5.50 ± 0.10ef 4.40 ± 0.10b 0.48 ± 0.03c 0.89 ± 0.040c 0.80 ± 0.020f 1.010 ± 0.020e 1.223 ± 0.020e 0.46 ± 0.041b 0.49 ± 0.04 b 0.25 ± 0.040a
16.67 ± 0.61j 13.97 ± 0.35i 11.93 ± 0.31h 1.88 ± 0.09e 0.75 ± 0.06e 0.55 ± 0.06c 2.84 ± 0.09g 3.03 ± 0.15f 0.99 ± 0.10b 0.53 ± 0.07b 0.43 ± 0.03b 0.47 ± 0.02d 6.67 ± 0.05bc 6.23 ± 0.21b 0.001 ± 0.00a 0.017 ± 0.006a 0.011 ± 0.001a
0.084 ± 0.004b 0.103 ± 0.007c 0.132 ± 0.002e 0.172 ± 0.003a 0.194 ± 0.002a 0.218 ± 0.002a 0.011 ± 0.001e 0.010 ± 0.001d 0.013 ± 0.001j 0.270 ± 0.002i 0.255 ± 0.003k 0.287 ± 0.004f 0.125 ± 0.003g 0.116 ± 0.006h 0.354 ± 0.006m 0.344 ± 0.006l 0.363 ± 0.006n
nd: not detected. Different letters (a–n) in the same columns are significantly different at the 5% level (p50.05).
Honeys Honey samples are collected from experienced beekeepers from Turkey in the 2014 harvest season. For characterizations of the honey varieties, melissopalynological analysis was performed following the method described by Louveaux et al.16. The dominant pollen percent of the samples is given in Table 1. The two varieties of honeys (oak and pine honeys) were honeydew honeys and their optic rotations were positive, in contrast to others were blossom17. Actually, identification of honeydew honey type was based on beekeepers’ declarations. Analyses of phenolic contents The polyphenolic contents of the methanolic samples were evaluated by three different ways: total phenolic contents (TPC), total flavonoids (TF) and condensed tannin (CT). Total phenolic compounds were determined by an end-point assay of Folin–Ciocalteu method18. The maximum absorbance was read at 760 nm and the results were calculated as mg of gallic acid equivalents per gram sample, using a standard graph. Total flavonoid contents of the honey samples were measured with a spectrophotometric method as reported previously19 using quercetin as standard. Briefly, 0.5 mL methanolic samples, 0.1 mL of 10% Al(NO3)3 and 0.1 mL of 1 M NH4.CH3COO were added to a test tube and incubated at room temperature for 40 min, then absorbance was measured against a blank at 415 nm20. The standard calibration curve was plotted using quercetin. The total flavonoid concentration was expressed as mg of quercetin equivalents per 100 g sample. Condensed tannins were determined according to the method of small modified Julkunen-Titto21. An aliquot (25 mL) of each extract or standard solution (tannic acids) was mixed with 750 mL of 4% vanillin (prepared with MeOH) and then 375 mL of conc. HCl was added. The well-mixed solution was incubated at room temperature in the dark for 20 min. The absorbance against blank was read at 500 nm. Tannic acid was used to make the standard curve (0.05–1 mg/mL). The results were expressed as mg tannic acid equivalents 100 g sample.
activity of each sample was determined using a slight modification of Sigma protocol and an actual reference method22. Briefly, the reaction mixture consisted of 100 mL of hyaluronidase (1.67 U/mg), 100 mL of phosphate buffer (200 mM, pH 7, 37 C) with 77 mM sodium chloride and 0.01% BSA mixed with 25 mL sample extract solution. After preincubation at 37 C for 10 min, the reaction was initiated by the addition 100 mL of substrate solution in the form of hyaluronoic acid (0.03% in 300 mM sodium phosphate, pH 5.35). The assay mixture was incubated at 37 C for 45 min. The undigested hyaluronoic acid was precipitated with 1 mL acid albumin solution made up of 0.1% bovine serum albumin in 24 mM sodium acetate and 79 mM acetic acid, pH 3.75. After leaving the mixture at room temperature for 10 min, the absorbance was measured at 600 nm using Thermo Scientific Evolution 260 spectrophotometer (Thermo Scientific, Waltham, MA). The assay was done in triplicate. The IC50 value was determined as the concentration of compound that gives 50% inhibition of maximal activity. To calculate the half maximal inhibitory concentration (IC50), a series of concentration and their response data are needed (e.g. sample concentrations X1, X2,. . ., Xn and % inhibition based on absorbance changing Y1, Y2,. . ., Yn). Percentage inhibition of the sample is calculated by the following equation: Inhibition percentage Mean of sample Asb 100 ¼ 100 Mean of control Asb The inhibition values of Y are in the range of 0–100. For concentration–response data, logarithmic function could be chosen instead of linear regression. After adding the response curve as logarithm transformed, X value as IC50 has to be calculated by the following equation: Y ¼ a lnðXÞ þ b Y ¼ 50 ! lnðXÞ ¼
50 b a
ð1Þ ð2Þ
In vitro anti-hyaluronidase activity assay
Results and discussion
UV spectroscopy technique was used to measure the inhibition of hyaluronidase and to detect its potential inhibitors. The inhibitory
This study determined the anti-inflammatory activities of various types of Turkish honeys in the form of anti-hyaluronidase activity.
Honey and BTHase
DOI: 10.3109/14756366.2015.1054819
100
Figure 1. The concentration-dependent antihyaluronidase activities of the honey samples.
3
Chestnut 1 Chestnut 2
90
Chestnut 3 Heather 1
80
Heather 2
% Inhibiton
70
Heather 3 Oak 1
60
Oak 2 Oak 3
50
Pine 1 40
Pine 2 Pine 3
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30
Buckwheat 1
20
Buckwheat 2
10
Heterofloral 2
Heterofloral 1 Heterofloral 3
0 0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration (g/mL)
Table 2. The calculated correlations among the studied parameters. Total phenolics Total phenolics Tannin Total flavonoids HYA
Pearson Sig. (p) Pearson Sig. (p) Pearson Sig. (p) Pearson Sig. (p)
correlation (r) correlation (r) correlation (r) correlation (r)
1 0.389* 0.005 0.585* 0.000 0.760* 0.000
Tannin 0.389* 0.005 1 0.773* 0.000 0.491* 0.000
Total flavonoids 0.585* 0.000 0.773* 0.000 1 0.755* 0.000
HYA 0.760* 0.000 0.491* 0.000 0.755* 0.000 1
*Correlation is significant at the 0.01 level.
The common names, floral sources and melissopalynological analysis of the honey samples are summarized in Table 1. This biological activity determined in the form of anti-hyaluronidase activity was observed to vary depending on the source of the nectar from which the honey was produced. Diluted extracts of 17 honeys from six different varieties were determined to produce inhibitory effects against hyaluronidase isolated from bovine testis from varying inhibition values (Figure 1). IC50 values ranged between 0.010 and 0.365 g/mL (Table 1). A low IC50 value from anti-hyaluronidase activity assay, a turbidimetric analysis technique, indicates high inhibition. The highest anti-hyaluronidase activity among the honeys in the study was exhibited by oak honey, followed by chestnut and buckwheat. Heterofloral blossom honeys and pine honeys exhibited the lowest inhibition effects on BTHase. Total polyphenol, total flavonoid and total tannin levels of ethanolic honey extracts were analyzed spectrophotometrically in order to reveal the secondary metabolites (Table 1). Total phenolic contents of the honeys ranged from 19 to 101 mg GAE/100 g. The ethanolic and phenolic contents were highest in heather, oak and chestnut honeys, while heterofloral blossom honeys and pine honeys exhibited the lowest levels. Our previous studies and other research have shown that oak, heather and chestnut honeys always exhibit higher phenolic compositions, as well as higher antioxidant capacities17,23–27. Polyphenols in honeys are responsible for sensory properties, such as odor, color, aroma and taste, and biologically active properties, such as antioxidant, antimicrobial, antitumoral and anti-inflammatory activities28. The phenolic substances are rather wide compounds
and contain many classes, such as phenolic acids, flavonols, flavonoids, procyanidins, anthocyanins and tannin29. The results of the study showed an important relation between honeys’ phenolic contents and anti-hyaluronidase activity (r2: 0.760, p50.01) (Table 2). Higher phenolic contents indicated greater inhibitory effects on BTHase. The highest level of inhibition was obtained from oak honeys. The characterization of honeydew honey differs from nectar honey. Oak honey is produced either from oak aphids, or else from sugary substance released from oak leaves under some stress conditions. Therefore, although both oak and pine honeys are honeydew characterized, oak honey has a rather different composition and biologically active properties17,30. The higher anti-hyaluronidase activity of oak honey may derive from its composition. In addition, oak honey either has a higher total phenolic content or else contains some specific phenolic agents. Our previous study showed that oak honey has higher levels of rutin, protocatechuic acid, gallic acid and catechin17. In addition to oak honeys, chestnut honey also exhibited high anti-hyaluronidase activity. In this study, chestnut honeys had the highest levels of condensed tannins and flavonoids. Other previous studies also reported that tannin-rich plant roots and stems possess high anti-hyaluronidase activity31,32. The highest level of tannin in this study was determined in chestnut honey and buckwheat honey. Total tannin levels of the two honeys were 18% and 12%, respectively. Similarly to chestnut honey, buckwheat honey also exhibited high anti-hyaluronidase activity, and this may derive from the high tannin levels in their structures. In addition, a statistically significant linear correlation was determined between honey’s total phenolic contents and
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anti-hyaluronidase activities (r2: 0.491, p50.01) (Table 2). In agreement with our study, previous in vitro research reported that 30 different flavonoid components exhibited inhibition against BTHase, and that inhibition levels were determined, from highest to lowest, in silibinin4kaempferol4apigenin4luteolin4tannin33. The idea that flavonoids possess anti-hyaluronidase activity has also been supported by other studies4,8,12,34. In conclusion, honey is a crucial inhibitory agent against hyaluronidase, the degrees of inhibition depending on the floral sources. Regular honey consumption may contribute in reducing inflammatory injury and strengthening human defense systems.
Acknowledgements We thank Dr. El-Sever ASADOV for the pollen analyses used in this study.
J Enzyme Inhib Med Chem, Early Online: 1–4
15.
16. 17. 18. 19. 20.
Declaration of interest The authors declare that they have no conflict of interests. We are grateful to TUBITAK (114Z370 projects) for supporting the study.
21. 22.
References 1. Meyer K. Hyaluronidases. In: Boyer PD, ed. The enzymes. New York: Academic Press; 1971:307–20. 2. Kaebler A, Olgen S, Jose J. Auto display of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors. Eur J Pharmacol Sci 2011;42:138–47. 3. Girish KS, Kemparaju K. The magic glue hyaluronan and its eraser hyaluronidase: a biological overview. Life Sci 2007;80:1921–43. 4. Sunitha K, Suresh P, Santhosh MS, et al. Inhibition of hyaluronidase by N-acetyl cysteine and glutathione: role of thiol group in hyaluronan protection. Int J Biol Macromol 2013;55:39–46. 5. Stern R. Hyaluronan metabolism: a major paradox in cancer biology. Pathol Biol 2005;53:372–82. 6. Stern R, Jedrzejas M. Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev 2006;106:818–39. 7. Stern R. Hyaluronidases in cancer biology. Semin Cancer Biol 2008; 18:275–80. 8. Selenge E, Murata T, Tanaka S, et al. Monoterpene glycosides, phenylpropanoids, and acacetin glycosides from Dracocephalum foetidum. Phytochemistry 2014;101:91–100. 9. Kaessler A, Nourrisson MR, Duflos M, Jose J. Indole carboxamides inhibit bovine testes hyaluronidase at pH 7.0 and indole acetamides activate the enzyme at pH 3.5 by different mechanisms. J Enzyme Inhib Med Chem 2008;23:719–27. 10. Wahby AF, Mahdy ESME, El-Mezayen HA, et al. Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom. Genet Eng Biotechnol J 2012;10:213–19. 11. Molander M, Nielsen L, Søgaard S, et al. Hyaluronidase, phospholipase A2 and protease inhibitory activity of plants used in traditional treatment of snakebite-induced tissue necrosis in Mali, DR Congo and South Africa. J Ethnopharmacol 2014;157:171–80. 12. Sobero´n J, Sgariglia MA, Sampietro DA, et al. Free radical scavenging activities and inhibition of inflammatory enzymes of phenolics isolated from Tripodanthus acutifolius. J Ethnopharmacol 2010;130:329–33. 13. Yıldız O, Karahalil F, Can Z, et al. Total monoamine oxidase (MAO) inhibition by chestnut honey, pollen and propolis. J Enzyme Inhib Med Chem 2014;29:690–4. 14. Sahin H, Aliyazıcıog˘lu R, Yıldız O, et al. Honey, polen, and propolis extracts show potent inhibitory activity against the zinc
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metalloenzyme carbonic anhydrase. J Enzyme Inhib Med Chem 2011;26:440–4. Sahin H, Can Z, Yildiz O, et al. Inhibition of carbonic anhydrase isozymes I and II with natural products extracted from plants, mushrooms and honey. J Enzyme Inhib Med Chem 2012;27: 395–402. Louveaux J, Maurizio A, Vorwohl G. Methods of melissopalynology. Bee World 1978;59:139–57. Can Z, Yıldız O, Sahin H, et al. An investigation of Turkish honeys; their physico-chemical properties, antioxidant capacities and phenolic profiles. Food Chem 2015;180:133–41. Singleton VL, Rossi JL. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am J Enol Viticult 1965;16:144–58. Fukumoto LR, Mazza G. Assessing antioxidant and prooxidant activities of phenolic compounds. J Agric Food Chem 2000;48: 3597–604. _ et al. Antioxidant activity and Bursal E, Ko¨ksal E, Gu¨lc¸in I, polyphenol content of cherry stem (Cerasus avium L.) determined by LC–MS/MS. Food Res Int 2013;51:66–74. Julkunen-Titto R. Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. J Agric Food Chem 1985;33:213–17. Yahaya YA, Don MM. Evaluation of Trametes lactinea extracts on the inhibition of hyaluronidase, lipoxygenase and xanthine oxidase activities in vitro. J Phy Sci 2012;23:1–15. Ku¨c¸u¨k M, Kolaylı S, Karaog˘lu S ¸ , et al. Biological activities and chemical composition of three honeys of different types from Anatolia. Food Chem 2007;100:526–34. ¨ ztu¨rk N, et al. Antioxidant activity and Sarıkaya AO, Ulusoy E, O phenolic acid content of chestnut honey and propolis. J Food Biochem 2009;33:470–81. Nasuti C, Gabbianelli R, Falcioni G, Cantalamessa F. Antioxidative and gastroprotective activities of anti-inflammatory formulations derived from chestnut honey in rats. Nutr Res 2006;26:130–7. Tezcan F, Kolaylı S, Sahin H, et al. Evaluation of organic acid, sacchraride composition and antioxidant properties of some authentic Turkish honeys. J Food Nutr Res 2011;50:33–40. Cavrar S, Yıldız O, Sahin H, et al. Comparison of physical and biochemical characteristics of different quality of Turkish honey. Uludag Bee J 2013;13:55–62. Kolayli S, Yildiz O, Sahin H, Aliyazıcıoglu R. Biochemistry and physicochemical properties of honey. In: Boukraa L, ed. Honey in traditional and modern medicine. Boca Raton (FL): CRC Press, Taylor & Francis Group; 2013:21–35. Havsteen BH. The biochemistry and medical significance of the flavonoids. Pharmacol Ther 2002;96:67–202. ¨ lgentu¨rk S, Szentkiralyi F, Uygun N, et al. Predators of Marchalina U hellenica (Hemiptera: Marchalinidae) on pine forest in Turkey. Phytoparasitica 2013;41:529–37. Piwowarskia JP, Kissa AK, Kozłowska-Wojciechowska M. Antihyaluronidase and anti-elastase activity screening of tannin-rich plant materials used in traditional Polish medicine for external treatment of diseases with inflammatory background. J Ethnopharmacol 2011;137:937–41. Ao C, Higa T, Ming H, et al. Isolation and identification of antioxidant and hyaluronidase inhibitory compounds from Ficus microcarpa L. fil. bark. J Enzyme Inhib Med Chem 2010;25: 406–13. Kuppusamy UR, Khoo HE, Das NP. Structure–activity studies of flavonoids as inhibitors of hyaluronidase. Biochem Pharmacol 2002; 40:397–401. Hertel W, Peschel G, Ozegowski JH, Mu¨ller PJ. Inhibitory effects of triterpenes and flavonoids on the enzymatic activity of hyaluronic acid splitting enzymes. Arch Pharm Chem Life Sci 2006;339: 313–18.
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RESEARCH ARTICLE
Honey shows potent inhibitory activity against the bovine testes hyaluronidase Sevgi Kolayli1, Huseyin Sahin2, Zehra Can3, Oktay Yildiz4, and Ku¨bra Sahin1 1
Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey, 2Espiye Vocational School, Giresun University, Espiye, Giresun, Turkey, 3¸Sebinkarahisar Technical Sciences Vocational School, Giresun University, Giresun, Turkey, and 4Mac¸ka Vocational School, Karadeniz Technical University, Mac¸ka, Trabzon, Turkey Abstract
Keywords
The purpose of this study was to investigate the anti-hyaluronidase activities of honeys from different botanical origins honeys in order to determine their anti-inflammatory properties. The total phenolic contents, total flavonoids and total tannin levels of six types of honey, chestnut, oak, heather, pine, buckwheat and mixed blossom, were determined. Concentrationrelated inhibition values were tested turbidimetrically on bovine testis hyaluronidase (BTHase) as IC50 (mg/mL). All honeys exhibited various concentration-dependent degrees of inhibition against BTHase. Inhibition values varied significantly depending on honeys’ levels of phenolic contents, flavonoid and tannin. The honeys with the highest anti-hyaluronidase activity were oak, chestnut and heather. In conclusion, polyphenol-rich honeys have high anti-hyaluronidase activity, and these honeys have high protective and complementary potential against hyaluronidase-induced anti-inflammatory failures.
Flavonoid, hyaluronidase, inhibition, phenolic content, tannin
Introduction Hyaluronidases are a class of enzymes which predominantly catalyze degradation of hyaluronic acid (HA) and are widespread in humans, bacteria and invertebrates1,2. HA or hyaluronan is a high molecular weight polysaccharide, the acidic glycosaminoglycan being composed of repeating disaccharide units of a-1,3 linked D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units2, does not contain sulfate groups, as well as heparin or chondroitin. The biopolymer is the main component of the extracellular matrix and connective tissues3,4. Concentration depends on the balance between HA syntheses via hyaluronate synthases and degradation via human hyaluronidases, mainly hyaluronidase 1 (hHyal-1), hyaluronidase 2 (hHyal-2) and PH-20 (hPH-20)5,6. The highly hydrophilic structure of HA means that it has many physiological roles, such as cell adhesion, migration and proliferation. Many studies have demonstrated the participation of HA in different physiological and pathophysiological conditions including embryogenesis, angiogenesis, wound healing, tissue turnover, malignancies and inflammatory disorders3,4. Hyaluronidases are involved in many pathological diseases, and their inhibitors serve as potential regulators as anti-inflammatory, anti-allergic, anti-tumoral, anti-aging, anti-rheumatoid, anti-toxin and antimicrobial agents4,7,8. The polysaccharide HA is important for tissue integrity, and has been reported to be associated with several diseases. HA fragments are reported to be Address for correspondence: Sevgi Kolayli, PhD, Department of Chemistry, Faculty of Science, Karadeniz Technical University, 61080 Trabzon, Turkey. Tel: +90 4623772487. Fax: +90 462325 3196. E-mail: [email protected]
History Received 21 March 2015 Revised 16 April 2015 Accepted 17 April 2015 Published online 15 June 2015
associated with cancer, and even to stimulate and facilitate cancer cell growth7,8. HA inhibitors have also led to new therapeutic concepts in pathophysiological conditions, such as laryngeal or breast cancer treatment, that are associated with the hyaluronan– hyaluronidase system3,4,7. Since the partial control of hyaluronidases possesses several important physiological functions, they have recently attracted considerable scientific interest as synthetic and natural inhibitors4,8–12. Honey and bee products are natural materials whose antioxidant, anti-inflammatory and anti-tumoral efficacies vary depending on the flora from which they are produced. Indeed, honey has been reported to have an inhibitory effect on monoamine oxidase13 and carbonic anhydrase14,15, and this is reported to derive more from honey polyphenols. No previous studies have investigated the anti-hyaluronidase activity of honey. This study for the first time investigated the anti-hyaluronidase activities of honey from various floral sources. The inhibition values obtained revealed a statistically significant association with honey’s phenolic contents.
Material and methods Reagents Bovine testes hyaluronidase (BTHase) (400–1000 U/mg solid), HAs odium salt and bovine serum albumin, sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic monohydrate (NaH2PO4H2O), gallic acid, sodium carbonate (Na2CO3), sodium acetate trihydrate (NaCH3COO3H2O) were purchased from Sigma-Aldrich (St. Louis, MO). Folin–Ciocalteu reagent was obtained from LiChrosolvÕ (Merck KGaA, Darmstadt, Germany).
2
S. Kolayli et al.
J Enzyme Inhib Med Chem, Early Online: 1–4
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Table 1. Identification markers of Turkish honeys and concentration of phenolic substances and anti-hyaluronidase activities (HYA).
Code
Honey samples
Predominant pollen (%)
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17
Chestnut Chestnut Chestnut Heather Heather Heather Oak Oak Oak Pine Pine Pine Buckwheat Buckwheat Heterofloral Heterofloral Heterofloral
76 71 68 92 83 80 nd nd nd nd nd nd 62 65 nd nd nd
Familia Castanea sativa Mill. Calluna vulgaris Quercus L. Pinusburia L. Fagopyrume sculentum – – –
Total phenolic (mg GAE/100 g)
Total flavonoids (mg QUE/100 g)
Total tannin (mg tannic acid/100 g)
HYA (IC50 g/mL)
92.28 ± 2.25k 83.10 ± 3.13j 67.56 ± 2.74h 101.67 ± 3.51ij 92.00 ± 3.00k 90.67 ± 2.08i 80.33 ± 1.52f 91.00 ± 1.00g 78.00 ± 2.65d 41.33 ± 0.57e 48.33 ± 2.52c 36.67 ± 2.51l 56.33 ± 1.52k 60.32 ± 1.53k 26.23 ± 0.75b 27.66 ± 0.67b 19.07 ± 0.68a
7.60 ± 0.10l 6.50 ± 0.15k 6.0 ± 0.10j 1.32 ± 0.08h 1.18 ± 0.07i 1.13 ± 0.06g 4.80 ± 0.20d 5.50 ± 0.10ef 4.40 ± 0.10b 0.48 ± 0.03c 0.89 ± 0.040c 0.80 ± 0.020f 1.010 ± 0.020e 1.223 ± 0.020e 0.46 ± 0.041b 0.49 ± 0.04 b 0.25 ± 0.040a
16.67 ± 0.61j 13.97 ± 0.35i 11.93 ± 0.31h 1.88 ± 0.09e 0.75 ± 0.06e 0.55 ± 0.06c 2.84 ± 0.09g 3.03 ± 0.15f 0.99 ± 0.10b 0.53 ± 0.07b 0.43 ± 0.03b 0.47 ± 0.02d 6.67 ± 0.05bc 6.23 ± 0.21b 0.001 ± 0.00a 0.017 ± 0.006a 0.011 ± 0.001a
0.084 ± 0.004b 0.103 ± 0.007c 0.132 ± 0.002e 0.172 ± 0.003a 0.194 ± 0.002a 0.218 ± 0.002a 0.011 ± 0.001e 0.010 ± 0.001d 0.013 ± 0.001j 0.270 ± 0.002i 0.255 ± 0.003k 0.287 ± 0.004f 0.125 ± 0.003g 0.116 ± 0.006h 0.354 ± 0.006m 0.344 ± 0.006l 0.363 ± 0.006n
nd: not detected. Different letters (a–n) in the same columns are significantly different at the 5% level (p50.05).
Honeys Honey samples are collected from experienced beekeepers from Turkey in the 2014 harvest season. For characterizations of the honey varieties, melissopalynological analysis was performed following the method described by Louveaux et al.16. The dominant pollen percent of the samples is given in Table 1. The two varieties of honeys (oak and pine honeys) were honeydew honeys and their optic rotations were positive, in contrast to others were blossom17. Actually, identification of honeydew honey type was based on beekeepers’ declarations. Analyses of phenolic contents The polyphenolic contents of the methanolic samples were evaluated by three different ways: total phenolic contents (TPC), total flavonoids (TF) and condensed tannin (CT). Total phenolic compounds were determined by an end-point assay of Folin–Ciocalteu method18. The maximum absorbance was read at 760 nm and the results were calculated as mg of gallic acid equivalents per gram sample, using a standard graph. Total flavonoid contents of the honey samples were measured with a spectrophotometric method as reported previously19 using quercetin as standard. Briefly, 0.5 mL methanolic samples, 0.1 mL of 10% Al(NO3)3 and 0.1 mL of 1 M NH4.CH3COO were added to a test tube and incubated at room temperature for 40 min, then absorbance was measured against a blank at 415 nm20. The standard calibration curve was plotted using quercetin. The total flavonoid concentration was expressed as mg of quercetin equivalents per 100 g sample. Condensed tannins were determined according to the method of small modified Julkunen-Titto21. An aliquot (25 mL) of each extract or standard solution (tannic acids) was mixed with 750 mL of 4% vanillin (prepared with MeOH) and then 375 mL of conc. HCl was added. The well-mixed solution was incubated at room temperature in the dark for 20 min. The absorbance against blank was read at 500 nm. Tannic acid was used to make the standard curve (0.05–1 mg/mL). The results were expressed as mg tannic acid equivalents 100 g sample.
activity of each sample was determined using a slight modification of Sigma protocol and an actual reference method22. Briefly, the reaction mixture consisted of 100 mL of hyaluronidase (1.67 U/mg), 100 mL of phosphate buffer (200 mM, pH 7, 37 C) with 77 mM sodium chloride and 0.01% BSA mixed with 25 mL sample extract solution. After preincubation at 37 C for 10 min, the reaction was initiated by the addition 100 mL of substrate solution in the form of hyaluronoic acid (0.03% in 300 mM sodium phosphate, pH 5.35). The assay mixture was incubated at 37 C for 45 min. The undigested hyaluronoic acid was precipitated with 1 mL acid albumin solution made up of 0.1% bovine serum albumin in 24 mM sodium acetate and 79 mM acetic acid, pH 3.75. After leaving the mixture at room temperature for 10 min, the absorbance was measured at 600 nm using Thermo Scientific Evolution 260 spectrophotometer (Thermo Scientific, Waltham, MA). The assay was done in triplicate. The IC50 value was determined as the concentration of compound that gives 50% inhibition of maximal activity. To calculate the half maximal inhibitory concentration (IC50), a series of concentration and their response data are needed (e.g. sample concentrations X1, X2,. . ., Xn and % inhibition based on absorbance changing Y1, Y2,. . ., Yn). Percentage inhibition of the sample is calculated by the following equation: Inhibition percentage Mean of sample Asb 100 ¼ 100 Mean of control Asb The inhibition values of Y are in the range of 0–100. For concentration–response data, logarithmic function could be chosen instead of linear regression. After adding the response curve as logarithm transformed, X value as IC50 has to be calculated by the following equation: Y ¼ a lnðXÞ þ b Y ¼ 50 ! lnðXÞ ¼
50 b a
ð1Þ ð2Þ
In vitro anti-hyaluronidase activity assay
Results and discussion
UV spectroscopy technique was used to measure the inhibition of hyaluronidase and to detect its potential inhibitors. The inhibitory
This study determined the anti-inflammatory activities of various types of Turkish honeys in the form of anti-hyaluronidase activity.
Honey and BTHase
DOI: 10.3109/14756366.2015.1054819
100
Figure 1. The concentration-dependent antihyaluronidase activities of the honey samples.
3
Chestnut 1 Chestnut 2
90
Chestnut 3 Heather 1
80
Heather 2
% Inhibiton
70
Heather 3 Oak 1
60
Oak 2 Oak 3
50
Pine 1 40
Pine 2 Pine 3
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30
Buckwheat 1
20
Buckwheat 2
10
Heterofloral 2
Heterofloral 1 Heterofloral 3
0 0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration (g/mL)
Table 2. The calculated correlations among the studied parameters. Total phenolics Total phenolics Tannin Total flavonoids HYA
Pearson Sig. (p) Pearson Sig. (p) Pearson Sig. (p) Pearson Sig. (p)
correlation (r) correlation (r) correlation (r) correlation (r)
1 0.389* 0.005 0.585* 0.000 0.760* 0.000
Tannin 0.389* 0.005 1 0.773* 0.000 0.491* 0.000
Total flavonoids 0.585* 0.000 0.773* 0.000 1 0.755* 0.000
HYA 0.760* 0.000 0.491* 0.000 0.755* 0.000 1
*Correlation is significant at the 0.01 level.
The common names, floral sources and melissopalynological analysis of the honey samples are summarized in Table 1. This biological activity determined in the form of anti-hyaluronidase activity was observed to vary depending on the source of the nectar from which the honey was produced. Diluted extracts of 17 honeys from six different varieties were determined to produce inhibitory effects against hyaluronidase isolated from bovine testis from varying inhibition values (Figure 1). IC50 values ranged between 0.010 and 0.365 g/mL (Table 1). A low IC50 value from anti-hyaluronidase activity assay, a turbidimetric analysis technique, indicates high inhibition. The highest anti-hyaluronidase activity among the honeys in the study was exhibited by oak honey, followed by chestnut and buckwheat. Heterofloral blossom honeys and pine honeys exhibited the lowest inhibition effects on BTHase. Total polyphenol, total flavonoid and total tannin levels of ethanolic honey extracts were analyzed spectrophotometrically in order to reveal the secondary metabolites (Table 1). Total phenolic contents of the honeys ranged from 19 to 101 mg GAE/100 g. The ethanolic and phenolic contents were highest in heather, oak and chestnut honeys, while heterofloral blossom honeys and pine honeys exhibited the lowest levels. Our previous studies and other research have shown that oak, heather and chestnut honeys always exhibit higher phenolic compositions, as well as higher antioxidant capacities17,23–27. Polyphenols in honeys are responsible for sensory properties, such as odor, color, aroma and taste, and biologically active properties, such as antioxidant, antimicrobial, antitumoral and anti-inflammatory activities28. The phenolic substances are rather wide compounds
and contain many classes, such as phenolic acids, flavonols, flavonoids, procyanidins, anthocyanins and tannin29. The results of the study showed an important relation between honeys’ phenolic contents and anti-hyaluronidase activity (r2: 0.760, p50.01) (Table 2). Higher phenolic contents indicated greater inhibitory effects on BTHase. The highest level of inhibition was obtained from oak honeys. The characterization of honeydew honey differs from nectar honey. Oak honey is produced either from oak aphids, or else from sugary substance released from oak leaves under some stress conditions. Therefore, although both oak and pine honeys are honeydew characterized, oak honey has a rather different composition and biologically active properties17,30. The higher anti-hyaluronidase activity of oak honey may derive from its composition. In addition, oak honey either has a higher total phenolic content or else contains some specific phenolic agents. Our previous study showed that oak honey has higher levels of rutin, protocatechuic acid, gallic acid and catechin17. In addition to oak honeys, chestnut honey also exhibited high anti-hyaluronidase activity. In this study, chestnut honeys had the highest levels of condensed tannins and flavonoids. Other previous studies also reported that tannin-rich plant roots and stems possess high anti-hyaluronidase activity31,32. The highest level of tannin in this study was determined in chestnut honey and buckwheat honey. Total tannin levels of the two honeys were 18% and 12%, respectively. Similarly to chestnut honey, buckwheat honey also exhibited high anti-hyaluronidase activity, and this may derive from the high tannin levels in their structures. In addition, a statistically significant linear correlation was determined between honey’s total phenolic contents and
4
S. Kolayli et al.
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anti-hyaluronidase activities (r2: 0.491, p50.01) (Table 2). In agreement with our study, previous in vitro research reported that 30 different flavonoid components exhibited inhibition against BTHase, and that inhibition levels were determined, from highest to lowest, in silibinin4kaempferol4apigenin4luteolin4tannin33. The idea that flavonoids possess anti-hyaluronidase activity has also been supported by other studies4,8,12,34. In conclusion, honey is a crucial inhibitory agent against hyaluronidase, the degrees of inhibition depending on the floral sources. Regular honey consumption may contribute in reducing inflammatory injury and strengthening human defense systems.
Acknowledgements We thank Dr. El-Sever ASADOV for the pollen analyses used in this study.
J Enzyme Inhib Med Chem, Early Online: 1–4
15.
16. 17. 18. 19. 20.
Declaration of interest The authors declare that they have no conflict of interests. We are grateful to TUBITAK (114Z370 projects) for supporting the study.
21. 22.
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