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UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA
DOCTORAL SCHOOL
FACULTY OF ANIMAL HUSBANDRY AND BIOTECHNOLOGIES
Eng. Cristina Manuela MIHAI
EVALUATION OF PROPOLIS QUALITY
FROM TRANSYLVANIA WITH
REGARDS TO STANDARDIZATION (SUMMARY OF PhD THESIS)
SCIENTIFIC COORDINATOR
Prof. Eng. Liviu Alexandru MĂRGHITA Ş, PhD
CLUJ-NAPOCA
2011
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CONTENTS
PhD page
Summary page
Introduction 11 28 Objectives 13 29
FIRST PART: LITERATURE REVIEW 14 CHAPTER I. PROPOLIS – GENERALITIES 15 30
1.1. Propolis importance 15 30 1.2. Chemical composition 17 30 1.3. Aspects regarding standardization 19
CHAPTER II. BIOLOGICAL ACTIVE COMPOUNDS FROM PROPOLIS: POLYPHENOLS 24 30
2.1. Phenolic acids 27 2.2. Flavonoids 27 2.3. Methods for polyphenolic compounds determination 29
2.3.1. Spectrophotometric methods 30 2.3.2. Chromatographic methods 32
CHAPTER III. THERAPEUTICAL EFFECTS OF PROPOLIS 34 3.1. Antioxidant capacity 35 3.2. Antimicrobial capacity 39 3.3. Other effects 40
CHAPTER IV. METHODS FOR THE DETERMINATION OF ANTIOXIDANT AND ANTIMICROBIAL CAPACITIES OF PROPOLIS 42
4.1. Methods for antioxidant capacity determination 42 4.1.1. Free radicals scavenging methods 43 4.1.2. Total antioxidant potential method (FRAP) 44
4.2. Methods for antimicrobial capacity determination 44 4.2.1. Diffusion method 45 4.2.2. Serial dilution method 46
SECOND PART: ORIGINAL RESEARCH 48 CHAPTER V. MATERIAL AND METHOD 49 31
5.1. Biologic material 49 31 5.2. Applied experimental methods 52 31
5.2.1. Alcoholic extracts preparation from propolis 52 5.2.2. Phisico –chemical analysis 54
5.2.2.1. Humidity determination 54 5.2.2.2. Wax content determination – Soxhlet method 55 5.2.2.3. Dry residue determination 56
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5.2.5. UV-Vis spectrofotometrical analysis 57 5.2.5.1. Total polyphenols content determination 57 5.2.5.2. Total flavonoid content determination 58 5.2.5.3. Antioxidant capacity determination through DPPH assay 62 5.2.5.4. Total antioxidant potential determination through FRAP assay 64
5.2.6. High performance liquid chromatography analysis (HPLC – PDA) 65
5.2.6.1. Propolis extracts preparation for HPLC – PDA analysis 65 5.2.6.2. HPLC – PDA cromatographic analysis 66
5.2.7. Microbiological analysis 72 5.2.7.1. Antimicrobial activity determination 72 5.2.7.2. Minimum inhibitory concentration (MIC) determination 73
5.3. Statistical analysis 74 CHAPTER VI. RESULTS REGARDING PHISICO – CHEMICAL PARAMETERS OF PROPOLIS 76 31
6.1. Results regarding water content (humidity) of propolis samples 76 6.2. Results regarding wax content of propolis samples 78 6.3. Results regarding dry residue of propolis alcoholic extracts 80 6.4. Discussions 81 6.5. Preliminary conclusions 93
CHAPTER VII. RESULTS REGARDING SPECTROPHOTOMETRIC DETERMINATIONS OF POLYPHENOLS, FLAVONOIDS AND ANTIOXIDANT CAPACITY OF PROPOLIS 95 34
7.1. Results regarding total polyphenols content from propolis 95 7.2. Results regarding total flavonoid content from propolis 97 7.3. Results regarding antioxidant capacity of propolis determined by DPPH method 102 7.4. Results regarding total antioxidant potential of propolis determined by FRAP method 104 7.5.Discussions 106 7.6.Correlations between phisico-chemical and spectrofotometrical analysis 136 7.7. Preliminary conclusions 142
CHAPTER VIII. REULTS REGARDING PHENOLIC COMPOUNDS OF PROPOLIS 144 39 8.1. Results regarding HPLC – PDA analysis of reference compounds 144 8.2. Results regarding HPLC – PDA analysis of propolis samples 147 8.3. Discussions 162
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8.4. Preliminary conclusions 170 CHAPTER IX. RESULTS REGARDING ANTIMICROBIAL CAPACITY OF PROPOLIS 172 40 9.1. Results regarding antimicrobial activity 172 9.2. Results regarding minimum inhibitory concentration (MIC) 180 9.3. Discussions 181 9.4. Correlations between microbiological analysis and phisico-chemical and spectrophotometrical analysis 9.5. Preliminary conclusions 196 CHAPTER X. GENERAL CONCLUSSIONS 199 43 ORIGINAL ELEMENTS 201 45 RECOMMENDATIONS AND PERSPECTIVES 201 45 LIST OF PUBLISHED PAPERS 202 BIBLIOGRAPHIC REFERENCES 204 45 APPENDIX 221 Appendix 1 – Abbreviations list 221 Appendix 2 – Calibration curves and UV-Vis spectra for reference compounds regarding HPLC – PDA anlysis 222 Appendix 3 – HPLC – PDA chromatograms for propolis samples 226 Appendix 4 – Antimicrobial activity – original photos 251 Romanian summary 257 English summary
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INTRODUCTION
Lately the interest regarding natural alternative of everything that is obtained
through synthesis is higher and higher. A large number of studies regarding propolis
show that this natural product obtained exclusively by bees and harvested by humans
possesses therapeutical effects. Among these effects are: antioxidant capacity,
antimicrobial capacity, hepatoprotective effect, antifungal activity, local anesthetic anti-
inflammatory effect and others.
Propolis is probably the most complex bee product, having popularity since
ancient times. Without a scientific base, our ancestors have used it as a remedy for
treatment of different diseases. Much more in our days, wehen chemical composition of
propolis is known and its terapeutical effects, we may trust using it.
Very often natural antioxidants are mentioned. Propolis is one of the most rich
natural product in antioxidants (flavonoids, phenolic acids and their esters). Along
antioxidant capacity, this product, propolis, shows a great antimicrobial activity, which is
supported by recent research studies. Due to these properties, propolis will become a
trusted substitute of antibiotics, which nowadays appear to have many side effects.
In the present PhD thesis, there is an important contribution regarding propolis
study by nodern chemical analysis, with the purpose of investigating Transylvanian
propolis quality. Quality control parameters are going to be proposed, which should be
checked when this product is used as a remedy. Propolis quality is determined by
biological active compounds content (phenolic acids and flavonoids), antioxidant
capacity, antimicrobial capacity and also polyphenolic profile. All analysis were
conducted in vitro under laboratory controlled conditions. Experimental procedures were
realized in the Bee Products Quality Control Laboratory, except microbiological
experiments which were conducted in the Microbiology Laboratory, both from
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, during
October 2008 – September 2011.
The present PhD thesis “Evaluation of propolis quality from Transylvania with
regards to standardization” is structured in two major parts. First one, “Literature
review” (Chapters I – IV) present the actual state of knowledge with regards to proposed
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objectives, like aspects concerning chemical composition, antioxidant and antimicrobial
capacities, methods used, and also the matter of standardization.
The second part “Original research” (Chapters V – X) presents used biological
material (53 propolis samples from Transylvania region), experimental procedures
(phisico-chemical, spectrophotometrical, chromatographic and microbiological), results
obtained, and general conclusions.
OBJECTIVES
Main objectives of the present PhD thesis are:
� Research regarding propolis composition, by determination of total amounts of
biological active principles (phenolic acids and flavonoids) by means of classical
spectrophotometric methods, and also wax content by authomatic Soxhlet
extraction.
� Research regarding antioxidant capacity of propolis from Transilvania (DPPH
method and FRAP method) evaluated in vitro by means of spectrophotometric
methods
� Research regarding antimicrobial capacity of propolis from Transilvania, by
testing it on internation reference bacterial strains: Staphylococcus aureus ATCC
6538P, Bacillus cereus ATCC 14579, Listeria monocitogenes ATCC 7644,
Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 27853, Candida
albicans ATCC 90028;
� Research regarding polyphenolic profile of propolis by High Performance Liquid
Chromatography (HPLC – PDA) with the purpose of identification and
quantification of specific compounds.
� Research regarding correlations between chemical composition and therapeutic
effects (antioxidant and antimicrobial capacities) of propolis.
� Indication of quality parameters of propolis which should be included when
proposing a standard.
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CHAPTER I. PROPOLIS – GENERALITIES
1.1. PROPOLIS IMPORTANCE
All bee products (honey, propolis, polen and royal jelly) show interes within
research teams, being products with a composition not much influenced by humans. All
these products posses therapeutical effects which are recognized worldwide.
1.2. CHEMICAL COMPOSITION
Propolis chemical composition is very variable, depending greatly of the flora
available to bees during harvesting (Marcucci, 1995; Bankova et al., 2000; Ahn and
colab et al., 2007). The interest of many research teams was and is to fully elucidate the
chemical composition of propolis. Until now more than 300 compounds from propolis
composition are known, from different categories: resins, wax, essential oils, minerals
and other substances (Burdock, 1998; Kosalec et al., 2003; Cunha et al., 2004;
Mărghitaş, 2005).
CHAPTER II. BIOLOGICAL ACTIVE COMPOUNDS FROM PROPOLIS:
POLYPHENOLS
Polyphenols are important antioxidants due to their high redox potential. Theirs
antioxidant capacity is considered to be much more higher than of essential vitamins,
having a significant contribution over therapeutic effects of products which contains them
(Tsao şi Deng, 2004). Among the role of antioxidants, polyphenols (especially
flavonoids) show antimicrobial activity (Boyanova şi colab., 2006). Due to therapeutic
effects of polyphenols, already proven by scientific studies, the interest for their
availability is higher and higher, being preferred natural sources.
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CHAPTER V. MATERIAL AND METHOD
5.1. BIOLOGIC MATERIAL
Biologic material subjected to the present study is constituted of 53 propolis
samples from Transylvania (figure 1). These samples were provided by beekeepers
during March 2009 – March 2010. All samples were kept in the freezer at -20˚C until
analysis. Samples were harvested by scraping propolis using hive tools according to
beekeepers.
Figure 1. propolis samples PS1-PS53 (original photo)
5.2. APPLIED EXPERIMENTAL METHODS
Experimental procedures were conducted using four types of analysis: physico-
chemical, spectrophotometric, chromatographic and microbiological.
CHAPTER VI. RESULTS REGARDING PHISICO – CHEMICAL PARAMETERS
OF PROPOLIS
Phisico-chemical analysis were used as a first indicator of propolis quality with
regards to water content, wax content and dry residue content.
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Water content or humidity of propolis samples was registered around
2.39±0.44%. The lowest value was determined for sample PS 45 (1.46±0.09%), while the
highest for sample PS 2 (3.39±0.11%).
Propolis samples distribution according to water content is shown in figure 2.
Samples show a normal distribution.
Figure 2. Histogram of propolis samples distribution according to their water
content
Wax content of propolis samples ranged between wide limits: 13.49±0.50% (PS
46) and 70.89±1.08% (PS 39), with the average of 34.20%. As for the propolis samples
distribution according to wax content, could be a normal one with the exception of three
samples with showed a very high content (PS 9, PS 30 and PS 39) (figure 3).
Dry residue content of propolis rabged between 37.11±0.35% (PS 30) and
98.06±0.35% (PS 19), with the average of 69.50%. According to this physico-chemical
parameter, two samples stand out with unexpected high values: PS 19 (98.06±0.35%) and
PS 13 (97.82±1.41).
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Figure 3. Histogram of propolis samples distribution according to their wax content
Dry residue content is a parameter with interest due to the fact that it reflects the
amount of extracted active principles from propolis by means of used extraction solvent.
Propolis samples distribution according to dry residue content is shown in figure 4.
Figure 4. Histogram of propolis samples distribution according to their dry residue
content
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CHAPTER VII. RESULTS REGARDING SPECTROPHOTOMETRIC
DETERMINATIONS OF POLYPHENOLS, FLAVONOIDS AND ANTIOXIDANT
CAPACITY OF PROPOLIS
Total polyphenols content of the 53 used propolis samples follows a normal
distribution as shown in figure 5.
Figure 5. Histogram of propolis samples distribution according to total polyphenols
content
The range of total polyphenols content was very wide, values were registered
between 23.25±3.20% (PS 12) and 63.23±2.44% (PS 41), with the average of 43.01%.
The two methods used for total flavonoid content show very different results as
shown in figures 6 and 7.
Antioxidant capacity of propolis was evaluated by means of two different
methods. The first one was DPPH method (radical scavenging activity), which offered
results in the range of 0.29±0.10 mmol Trolox / g propolis (PS 30) and 1.40±0.10 mmol
Trolox / g propolis (PS 19). The second one was FRAP method (Ferric reducing
antioxidant potential). „ FRAP values” obtained for propolis samples were in the range of
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0.57±0.04 mmol FeSO4 / g propolis (PS 17) and 2.55±0.10 mmol FeSO4 / g propolis (PS
29) (figures 8 and 9).
Figure 6. Total flavonoid content determined by the two methods applied
(samples PS 1 – PS 25)
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Figure 7. Total flavonoid content determined by the two methods applied
(samples PS 26 – PS 53)
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Figure 8. Comparative study of the two applied antioxidant methods
(samples PS 1 – PS 25)
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Figure 9. Comparative study of the two applied antioxidant methods
(samples PS 26 – PS 53)
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CHAPTER VIII. RESULTS REGARDING PHENOLIC COMPOUNDS OF
PROPOLIS
All propolis samples were subjected to HPLC-PDA analysis, chromatograms were
registered at 280 nm and 340 nm, wavelengths specific to phenolic acids and flavonoids.
UV-Vis spectra corresponding to each signal from chromatograms were recorded in the
range 190-650 nm. Two of the propolis samples outstanded due to identification od 10
reference compounds in theirs chemical composition (figures 10 and 11).
10 20 30 40 50
0
400000
800000
1200000
Abs
orba
nta
[mA
U]
Abso
rbance
[m
AU
]
PS 22
Timp de retentie [min]Retention time [min]
Acid si
ringic
/13,61
3
Acid ca
feic/
14,9
63
Vanilin
a/16,
585
Acid p
-cum
aric/
20,2
14
Acid fe
rulic/
22,0
19
Acid t-
cinam
ic/32
,960
Pino
cembr
ina/4
2,176
Crisina
/43,90
7
Galang
ina/
44,9
30
Pino
strob
ina/47
,980
Figure 10. HPLC – PDA chromatogram of propolis sample PS 22
Both samples, were harvested in Mureş county (PS 22 and PS 24), being the only
ones in which t-cinnamic acid was cuantified in relatively low concentrations, of 0.64
and 0.71 mg t-cinnamic acid / g propolis. Among reference compounds identified in
studied propolis samples, three (caffeic acid, p-coumaric acid and ferrulic acid) were
present in all samples. Flavonoids used as reference compounds for spectrophotometric
determinations resulted to be appropriate according to HPLC – PDA analysis.
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10 20 30 40 50
0
400000
800000
1200000
PS 24
Abs
orba
nta
[mA
U]
Ab
sorb
anc
e [
mA
U]
Timp de retentie [min]Retention time [min]
Acid s
iring
ic/13
,656
Acid c
afeic/
15,02
3
Vanilin
a/16,4
48
Acid p-
cum
aric/
20,28
6
Acid fe
rulic/
22,0
76
Acid t-
cinam
ic/33
,008
Pino
cem
brina/
41,9
45
Crisin
a/43,9
49
Galan
gina/4
4,98
7
Pinostr
obina
/48,0
02
Figure 11. HPLC – PDA chromatogram of propolis sample PS 24
Pinocembrin, the flavanone used for flavanones/ dihydroflavonols content
determination was identified and cuantified in 39 propolis samples, the concentration
range being 0.03 ÷ 2.85 mg / g propolis, with an average of 1.05 mg / g propolis. Among
identified flavonoids in propolis, chrysin was the flavone most aboundant. The
concentration range for chrysin was 0.07 ÷ 3.91 mg / g propolis, with an average of 1.59
mg / g propolis. Regarding the flavonols galangin, it was cuantified in 34 propolis
samples, in the concentration range of 0.31 ÷ 3.20 mg / g propolis, with an average of
1.39 mg / g propolis.
CAPITOLUL IX. RESULTS REGARDING ANTIMICROBIAL CAPACITY OF
PROPOLIS
Antimicrobial activity was investigated on six different bacterial strains as
mentiond in chapter “Material and method”. Propolis samples distribution according to
inhibition zone diameter which represent the antimicrobial activity (Staphylococcus
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aureus, Bacillus cereus, Listeria monocitogenes, Escherichia coli and Candida albicans)
is shown in figures 12 – 16.
Regarding gram-negative bacteria Pseudomonas aeruginosa, samples distribution
is not shown due to the resistance it presented to all tested alcoholic propolis extracts.
Figure 12. Histogram of propolis samples distribution according to antimicrobial activity
against Staphylococcus aureus
Figure 13. Histogram of propolis samples distribution according to antimicrobial activity
against Bacillus cereus
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Figure 14. Histogram of propolis samples distribution according to antimicrobial activity
against Listeria monocitogenes
Figure 15. Histogram of propolis samples distribution according to antimicrobial activity
against Escherichia coli
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Figure 16. Histogram of propolis samples distribution according to antimicrobial activity
against Candida albicans
CHAPTER X. GENERAL CONCLUSSIONS
According to proposed objectives of the present PhD thesis, the following could
be concluded:
1. Chemical composition of propolis from Transylvania was determined, by
evaluation of total amounts of biologic active principles (polyphenols and
flavonoids), wax content, water content and dry residue content.
2. Antioxidant capacity of propolis was determined by means of two
spectrophotometric methods: radical scavenging activity (DPPH method) and total
antioxidant potential (FRAP method). Both methods confirm that propolis
possesses antioxidant capacity.
3. Antimicrobial capacity of propolis against six bacterial strains was determined:
Staphylococcus aureus ATCC 6538P, Bacillus cereus ATCC 14579, Listeria
monocitogenes ATCC 7644, Escherichia coli ATCC 10536, Pseudomonas
aeruginosa ATCC 27853, Candida albicans ATCC 90028. All propolis samples
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showed in vitro antimicrobial activity against Gram-positive bacteria and selective
activity against Gram-negative bacteria.
4. Polyphenolic profile of propolis was determined by means of HPLC – PDA
chromatographic technique, which was as follows: syringic acid, caffeic acid,
vanillin, p-coumaric acid, ferrulic acid, t-cinnamic acid, pinocembrin, chrysin,
galangin and pinostrobin. The presence of pinocembrin, chrysin, galangin and
caffeic acid in the polyphenolic profile of propolis allowed us to classify it as
poplar propolis.
5. Correlations between chemical composition and studied therapeutic effects were
determined. Flavonoid content correlates in a high manner with antioxidant
capacity of propolis. Flavones/flavonols and flavanones/dihydroflavonols are
responsible for antimicrobial activity of propolis as shown by Pearson correlation
coefficients. Total polyphenols concentration can not be considered a major
criteria when estimating antioxidant and antimicrobial capacities, due to weak
correlations shown between these parameters. Wax content could be used as a first
indicator of propolis quality. A high content of wax correlates with a low content
of active principles (flavonoids and phenolic acids).
6. Parameters that should be considered for quality control of propolis are:
a. Water content – maximum 10%;
b. Dry residue content – minimum 35%;
c. Wax content – maximum 35%;
d. Total flavonoids content – minimum 10%;
e. Total polyphenols content – minimum 20%;
f. Antioxidant capacity;
g. Antimicrobial capacity.
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ORIGINAL ELEMENTS
1. Polyphenolic profile characterization of propolis from Transylvania by means of
chromatographic technique (HPLC – PDA) ant its classification as poplar
propolis.
2. Evaluation of antimicrobial capacity of propolis against six international bacterial
strains.
3. Indication of quality control parameters (wax content, water content, dry residue
content, total polyphenols, total flavonoids, antimicrobial and antioxidant
activities) necessary to be checked before its use as therapeutical agent.
RECOMMENDATIONS AND PERSPECTIVES
1. Extension of the present study on country level and standard proposal requiring
propolis quality parameters.
2. Wax content and total flavonoid content should be checked as first indicators of
propolis quality.
3. Use of propolis for therapeutical pourposses only after quality control.
BIBLIOGRAPHIC REFERENCES
1. Ahn, M. R., S. Kumazawa, Y. Usui, J. Nakamura, M. Matsuka, F. Zhu, T. Nakayama, 2007, Antioxidant activity and constituents of propolis collected in various areas of China, Food Chemistry, 101, 1383-1392.
2. Bankova Vassya, S. L. De Castro, M. C. Marcucci, 2000, Propolis: recent advances in chemistry and plant origin, Apidologie, 31, 3-15.
3. Boyanova Lyudmila, R. Kolarov, Galina Gergova, I. Mitov, 2006, In vitro activity of Bulgarian propolis against 94 clinical isolates of anaerobic bacteria, Anaerobe, 12, 173-177.
4. Burdock, G. A., 1998, Review of the biological properties and toxicity of bee propolis (propolis), Food and Chemical Toxicology 36, 347-363.
5. Cunha, I. B. S., A. C. H. F. Sawaya, F. M. Caetano, M. T. Shimiziu, M. C. Marcucci, F. T. Drezza, G. S. Povia, P. O. Carvalho, 2004, Factors that Influence
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the Yield and Composition of Brazilian Propolis Extracts, Journal of Brazilian Chemical Society, 15:6, 1-xxx.
6. Kosalec, I., Marina Bakmaz, S. Pepeljnjak, 2003, Analysis of propolis from the continental and Adriatic regions of Croatia, Acta Pharmaceutica, 53, 275-285.
7. Marcucci, M., 1995, Propolis, chemical composition, biological properties and therapeutic activity, Apidologie 26, 83-99.
8. Mărghitaş, L. Al. , 2005, Albinele şi produsele lor, ediţia 2, Ed. Ceres, Bucureşti. 9. Tsao, R., Z. Deng, 2004, Separation procedures for naturally occurring antioxidant
phytochemicals. Journal of Chromatography B, 812, 85-99.
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