research article application of cornelian cherry iridoid...

Research ArticleApplication of Cornelian Cherry Iridoid-Polyphenolic Fractionand Loganic Acid to Reduce Intraocular Pressure

Dorota Szumny12 Tomasz SozaNski1 Alicja Z Kucharska3

Wojciech Dziewiszek1 Narcyz Pioacuterecki45 Jan Magdalan1 Ewa Chlebda-Sieragowska1

Robert Kupczynski6 Adam Szeldg1 and Antoni Szumny7

1Department of Pharmacology Wrocław Medical University 50-345 Wrocław Poland2Ophthalmology Clinic Uniwersytecki Szpital Kliniczny 50-556 Wrocław Poland3Department of Fruit and Vegetables Technology Wrocław University of Environmental and Life Sciences 51-630 Wrocław Poland4Arboretum and Institute of Physiography in Bolestraszyce 37-722 Bolestraszyce Poland5Department of Turism amp Recreation University of Rzeszow 35-959 Rzeszow Poland6Department of Environment Hygiene and Animal Welfare Wrocław University of Environmental and Life Sciences51-630 Wrocław Poland7Department of Chemistry Wrocław University of Environmental and Life Science 50-375 Wrocław Poland

Correspondence should be addressed to Dorota Szumny dorotaszumnywppl

Received 6 February 2015 Revised 21 April 2015 Accepted 12 May 2015

Academic Editor MinKyun Na

Copyright copy 2015 Dorota Szumny et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

One of the most common diseases of old age in modern societies is glaucoma It is strongly connected with increased intraocularpressure (IOP) and could permanently damage vision in the affected eye As there are only a limited number of chemical compoundsthat can decrease IOP as well as blood flow in eye vessels the up-to-date investigation of newmolecules is importantThe chemicalcomposition of the dried Cornelian cherry (Cornus mas L) polar iridoid-polyphenol-rich fraction was investigated Loganic acid(50) and pelargonidin-3-galactoside (7) were found as the main components Among the other constituents iridoid compoundcornuside and the anthocyans cyanidin 3-O-galactoside cyanidin 3-O-robinobioside and pelargonidin 3-O-robinobioside werequantified in the fraction In an animal model (New Zealand rabbits) the influence of loganic acid and the polyphenolic fractionisolated from Cornelian cherry fruit was investigated We found a strong IOP-hypotensive effect for a 07 solution of loganic acidwhich could be compared with the widely ophthalmologically used timolol About a 25 decrease in IOP was observed within thefirst 3 hours of use

1 Introduction

The term ldquoglaucomardquo involves a range of diseases associatedwith progressive optic nerve damage including typical mor-phological changes of its disk and characteristic loss of thefield of vision which are often accompanied by increasedintraocular pressure (IOP) [1] Risk factors of glaucoma-associated optic nerve damage include increased IOP positivefamily history systemic arterial hypotension or hypertensionand ischaemia-aggravating diseases such as diabetes [2 3]

The maintenance of IOP at a desired level is particu-larly important in the therapy of glaucoma [4] Results of

published studies demonstrate that a reduction in IOP isassociated with a lower risk of glaucoma progression owingto the less rapid optic nerve damage and reduced loss ofthe visual field [5ndash7] Pressure reduction offered by thecurrently used medication is based on reduced productionor increased outflow of aqueous media [8] some of whichalso have neuroprotective properties or are able to improvevascular flow Nowadays the following groups of medicationare used for glaucoma treatment 120573-adrenergic receptorantagonists prostaglandin analogues 120572

2-adrenergic receptor

agonists carbonic anhydrase inhibitors cholinergic agentsand hyperosmotic drugs [9 10]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 939402 8 pageshttpdxdoiorg1011552015939402

2 Evidence-Based Complementary and Alternative Medicine

Only a few studies have been aimed at the effect ofcompounds of natural origin on the IOP as well as otherglaucoma-progressing factors such as vascular blood flowantioxidative effects or neuroprotection The majority ofthese studies were conducted in vitro but a few were invivo and reached clinical trials The difficulties encounteredin the evaluation of natural compounds on the progress ofglaucoma-associated neuropathy are a consequence of long-term disease development but are also because of the limitedpossibility to use proper animal models [11 12]

One of several plant-origin compounds with a provenIOP-reducing effect is forskolin which is obtained fromColeus forskohlii (Lamiaceae) Following intraconjunctivaladministration the compound stimulates adenylate cyclasewhich leads to the activation of cyclic adenosine monophos-phate in ciliary epithelium and the reduction of IOP resultingfrom reduced production of aqueous products in rabbitsmonkeys and humans [13 14] Similarly natural bromophe-nols isolated from red algae belonging to the Rhodomelaceaefamily such as vidalol B present activity as carbonic anhy-drase inhibitors and may become a valuable IOP-reducingdrug in the future [15] Recently marijuana was consideredas an IOP-reducing natural compoundHowever because of ahigh ratio of nonresponders short half-life and considerabletoxicity the substance was not qualified for medicinal use[16ndash18] Among other plants Ginkgo biloba leaf extracts maybe used for adjunct glaucoma therapy The extract containspolyphenolic flavonoid compounds as well as terpenoidsand is able to scavenge free radicals at themitochondrial levelin order to improve blood flow In addition the substanceprevents neurotoxicity of glutamate whichmay be importantfor a neuroprotective effect and inhibits lipid peroxidation[19 20] Although it does not affect the IOP it may improvevascular blood flow into the optic nerve [21] Clinical trialshave indicated that anthocyanins and Ginkgo biloba extractmay be effective in improving the visual function and thevisual field of patients with normal-tension glaucoma [2022] Nowadays the only drug of natural origin currentlyused in glaucoma treatment is pilocarpine which is thealkaloid from Jaborandi (Pilocarpus microphyllus) leaves Itspharmacological activities are connected with properties ofa muscarinic agonist with no nicotinic effects [23 24] Oneof the most popular plant extracts traditionally used in eyesdiseases in Europe especially for the inflammation of eyesis Eyebright (Euphrasia) Its most important constituentsthat affect the eyes are iridoid glycosides including aucubingeniposide catalpol and luproside [25ndash27] Although thereare no relevant clinical studies an established traditional useof those eye drops proves their efficacy in conjunctivitis [28]

To the best of our knowledge there is a lack of dataregarding the effect of loganic acid or Cornelian cherrypreparations on IOP or vascular flow in the eye We havenot found any loganic-acid-containing or Cornelian-cherry-based preparations registered in the world which couldbe used for the therapy of glaucoma reduction of IOP orincreasing vascular blood flow in the eye

The aim of our study was to investigate the influence ofCornus mas iridoid-rich extract as well as its predominantcompound loganic acid on IOP

2 Materials and Methods

21 Evaluation of Cornelian Cherry Constituents Raciborski-variety Cornelian cherries (Cornus mas L) were obtainedfrom the Bolestraszyce Arboretum and Institute of Phys-iography Poland in September 2012 The plant materialwas authenticated by Professor Jakub Dolatowski and thevoucher specimen (12402) was deposited at the Herbariumof Arboretum and Institute of Physiography in BolestraszycePoland The polyphenolic and loganic acid iridoid fractionswere prepared in the Department of Fruit Vegetable andCereals Technology at Wroclaw University of Environmentaland Life Science according to the procedure described inour previous publication [29] and patent [29] Ripe Cor-nelian cherries were washed frozen and stored at minus20∘Cuntil processing After freezing removing stones defrostingand depectinisation we obtained the juice The juice waspurified on an Amberlite XAD-16 resin column Iridoidsand phenolics were eluted with 80 ethanol The eluent wasconcentrated under vacuum at 40∘C and lyophilised About20 g of lyophilisate from the iridoid-polyphenolic fractionwas obtained from 5 kg of fruit After freeze drying thesamples were ground into a powder using a laboratory millThe structure of the compounds in the iridoid-phenolicfraction was determined by UPLC-MS analysis

22 Isolation of Anthocyan-Irdoid Fraction and Pure LoganicAcid The easy effective and low-cost approach for isolationof loganic acid as well as the anthocyanin fraction wasdeveloped in our research group [30] In brief freeze-diedCornelian fruit powder (as described above) was dissolvedin 30 ethanol and separated using column chromatography(polyamide) Elution with 50 ethanol (and 1 acetic acid)afforded the anthocyanin fraction and pure loganic acidApplying the described procedure to 1 kg of fresh fruitallowed us to obtain 40 g of the anthocyanin fraction and15 g of pure loganic acid

The structure and purity of loganic acid was determinedby NMR and UPLC-MS analyses

23 Identification of Compounds by UPLC-MSMS andHPLCCompounds of the phenolic-iridoid and loganic acid frac-tions were identified by applying the method described byKucharska et al [30] using the acuity ultra-performanceliquid chromatography (UPLC) system coupled with aquadrupole time-of-flight (Q-TOF) MS instrument (WatersCorp Milford MA USA) with an electrospray ionisation(ESI) source Separation was achieved on the Acquity BEHC18 column (100mm times 21mm id 17mm Waters) Detec-tion wavelengths were set to 245 and 520 nm The mobilephase was a mixture of 45 formic acid (A) and acetonitrile(B) The gradient program was as follows initial conditions99 (A) 12min 75 (A) 125min 100 (B) 135min 99(A)The flow rate was 045mLmin and the injection volumewas 5 120583L The column was operated at 30∘C The instrumentwas operated in both positive- (anthocyanins) and negative-ion (iridoids) modes scanning the mz from 100 to 1500 ata scan rate of 20 scycle The major operating parameters for

Evidence-Based Complementary and Alternative Medicine 3

the Q-TOF MS were set as follows capillary voltage 20 kVcone voltage 45V cone gas flow of 11 Lh collision energy28 eV source temperature 100∘C desolvation temperature250∘C collision gas argon and desolvation gas (nitrogen)flow rate 600 Lh The data were collected with Mass-Lynx V41 software

Additionally compounds in the obtained phenolic-iridoid and loganic acid fractions were quantified usingthe Dionex HPLC system (Germering Germany) equippedwith the Ultimate 3000 model diode-array detector Thedetector cooperated with the LPG-3400A pump EWPS-3000SI autosampler column thermostat TCC-3000SD andChromeleon version 68 software A Cadenza Imtakt C5ndashC18 column (75 times 46mm) was used with a guard columnThe mobile phase was composed of solvent A (45 formicacid vv) and solvent B (acetonitrile)The elution system wasas follows 0-1min 5 B 20min 25 B 21min 100 B26min 100 B 27min 5 B The flow rate of the mobilephase was 10mLmin and the injection volume was 20 120583LThe column was operated at 30∘C The runs were monitoredat wavelengths of 245 (iridoids) and 520 nm (anthocyanins)Iridoids were quantified as loganic acid and anthocyaninsas cyanidin 3-O-glucoside The results were calculated asmilligrams of compound in 1 g dryweight ofCornelian cherrypowder (mgg dw) All determinations were performed intriplicate

24 NMR Analysis The NMR spectrum of loganic acid wasmeasured on an NMR Bruker AVANCE 600MHz spectrom-eter in the Laboratory of Structural Analysis at the TechnicalUniversity of Wrocław The chemical structure was estab-lished on the basis of 1H and 13C NMR as well as correlativeHSQC HMBC and ROESY spectra The measurement wasperformed at 24∘C

25 Reference Material Themaleate salt of timolol (1 conceye-drops) was purchased as commercially available OftanTimolol (Santen Poland) and used as in the reference

26 Biological Assays For the evaluation of the effect ofloganic acid or the polyphenolic fraction on IOP and bloodflow in blood vessels of the iris 14 young sexually matureNew Zealand white rabbits aged between 6 and 12 monthswere used 7 males and 7 females During the experiment therabbits were kept in individual cages at 21ndash23∘CThe animalswere fed with a full-meal blend rabbit feed (special blend forthe time of biological testing LSK Agropol SJ) ad libitumwith unlimited access to drinking water

Before the experiment itself the animals were kept under4-week quarantine The behaviour of the animals and theirphysical condition were observed during that time Themeasured data throughout all experiments were obtained byone person in order to achieve domestication and to reducethe amount of stress which could interfere with results of theexperiment

A 07 solution of loganic acid or the polyphenolic frac-tion was administered with an aqueous solution of artificialtears containing 015 sodium hyaluronate (vehicle)

In the first part of the study each animal was given 07loganic acid or polyphenolic fraction by intraconjunctivaladministration of one drop corresponding to a volume of50 120583L to the right eye (study group) whereas the vehicle inone drop (50120583L) was administered to the left eye as a placebo(control group) Then the IOP was measured in both eyesat six time points before administration and 1 2 3 4 and5 hours after administration of loganic acid and the vehicleInduction tonometer Icare requiring no prior anaesthesiawas used for the measurement of the IOP Reproducibility ofthe data has widely been described by other authors [31ndash34]

In the second part of the study each animal was given07 loganic acid through intraconjunctival administrationin one drop (50120583L) to the right eye whereas the vehicle wasagain administrated to the left eye in one drop (50120583L) Bloodflow in the iris was measured before administration and 12 3 4 and 5 hours after administration The measurementwas completed using a laser Doppler flowmeter (Laser BloodFlow Monitor MBD

3 Moor Instruments GB) Following

local anaesthesia of the cornea (Alcaine Alcon) the laser wasplaced in direct contact with the cornea (ca 2mm from thecorneal limbus)The laser beamwas directed perpendicularlyto the surface of the iris The measurement time was 50seconds During that time the apparatus performed 500measurements of momentary capillary flow

The interval between the first and the second parts of thedescribed experiments was 2 days

27 Statistical Analysis The following tests were used todetermine differences between groups the nonparametricWilcoxon test was used for comparison of IOP values andStudentrsquos t-test was used for comparison of iris blood flowvalues

3 Results and Discussion

The easy effective and low-cost approach for the isolationof loganic acid as well as the anthocyanin fraction wasdeveloped in our research group and led to the isolation ofbiologically active compounds

The chemical composition of the polar fraction obtainedfrom Cornelian cherry fruit is presented in Table 1 The pre-dominant compounds were iridoids loganic acid togetherwith cornuside (about 55) in the mixture Of the antho-cyanins cyanidin and pelargonidin-3-galactoside prevailedAmong the other constituents the iridoid compoundcornuside and the following anthocyans cyanidin 3-O-galactoside cyanidin 3-O-robinobioside and pelargonidin 3-O-robinobioside were quantified in fraction

Contrary to our results the recent study by Pawlowskaet al [35] shows pelargonidin-3-glucoside together withcyanidin galactoside as the main flavonoids The iridoid andflavonoid fraction of C mas is quite different to the C offici-nalis traditionally used in Chinese medicine to increase urineflow and decrease blood pressure [36]Themain constituentsof the iridoid fraction of C mas fruits were loganic acidand morroniside which are considered as biologically activecompounds [37]


Table 1 The chemical composition of the iridoid-polyphenolic fraction of Cornelian cherries (Raciborski variety)

Compound [M minusH]minus[M + H]+(mz) Ionisation (mgg)

IridoidsLoganic acid (LA) 3751276 minus 21669 plusmn 425 5024Cornuside (Co) 5411566 minus 2155 plusmn 029 500Total 23824

AnthocyaninsDelphinidin 3-O-galactoside (Df-gal) 4651034 + 063 plusmn 002 015Cyanidin 3-O-galactoside (Cy-gal) 4491063 + 1579 plusmn 026 366Cyanidin 3-O-robinobioside (Cy-rob) 5951713 + 638 plusmn 004 148Pelargonidin 3-O-galactoside (Pg-gal) 4331125 + 2994 plusmn 024 694Pelargonidin 3-O-robinobioside (Pg-rob) 5791766 + 595 plusmn 008 138Total 5869

FlavonolsQuercetin 3-O-glucuronide (Q-glucr) 4770665 minus 486 plusmn 017 113Kaempferol 3-O-galactoside (Kf-gal) 4470916 minus 403 plusmn 020 093Total 889

Phenolic acids3-O-Caffeoylquinic acid (3-CQA) 3530879 minus 347 plusmn 008 0815-O-Caffeoylquinic acid (5-CQA) 3530879 minus 1089 plusmn 019 252Total 1436

The structure of loganic acid (Figure 1) was confirmed byNMR obtaining appropriate chemical-shift values 13CNMR(DMSO-D

6) 120575 (ppm) 16856 -CO

2H 15055 C-3 11305 C-4

9897 C-11015840 9648 C-1 7766 C-51015840 7722 C-31015840 7361 C-21015840 7264C-7 7054 C-41015840 6160 C-61015840 4520 C-9 4221 C-6 4097 C-8 3134 C-5 1401 C-10 1H NMR (DMSO-D

6) 120575 (ppm) 729

(1H s H-3) 509 (1H d J = 49Hz H-1) 448 (1H d J =79Hz H-11015840) 388 (1H t J = 47Hz) 366 (1H d J = 117HzH-61015840a) 344 (1H dd J = 117 and 64Hz H-61015840b) 315 (1H dd J= 90 and 86Hz) 314 (1H m H-51015840) 305 (1H dd J = 94 and90Hz H-41015840) 297 (1H t J = 86Hz H-31015840) 292 (1H q J =79Hz H-5) 207 (1H dd J = 132 and 79Hz H-6) 181 (1Hddd J = 86 79 and 49Hz H-9) 171 (1H m H-8) 144 (1Hddd J = 132 79 and 47Hz H-6) 098 (3H d J = 68HzCH3-) The chemical structure of the analysed compound

was typical for iridoids and the diagnostic for interpretationsignals was the ether enolic proton (C-3) at 729 ppm with acarbon at 15055 ppm correlated in the HMBC spectrum toacetal H-1 proton and carboxyl group present in the loganicacid structure Additionally a sugar moiety coupled to theiridoid was identified according to the available 13C shiftsdatabase in [38] The obtained spectra (chemical shifts ofprotons and carbon atoms) were consistent with previouslyreported data [39 40] and supported the results of the HPLCand MS ([M minus H]minus ion at mz 375) analyses However thisis the first spectrum of loganic acid recorded in deuterateddimethyl sulfoxide

The presence of loganic acid (Figure 1) and cornuside inC mas fruits was also proven by Deng et al [41] but on thebasis of UV spectroscopy and the molecular mass The sameauthors identified loganin (methyl ester of loganic acid) as

O

O

OOH

OH

OH

OH

OH

O

HO

H

HH3C

Figure 1 Structure of loganic acid

well as sweroside although they did not perform quantitativeanalyses Loganic acid belongs to a large natural-compoundgroup of terpenes called iridoids which has also been foundin plants like Gentiana linearis [42] Strychnos nux-vomica[40] or recently Cornus officinalis fruits [37 43] but inmuch lower concentrations The related C mass species Cofficinalis grown in Asia has a strong beneficial effect ondiabetic nephropathy [44] Its activity was proven both in ananimal model [45] and on the basis of traditional Chinesemedicine [46] According to Jiang this activity is stronglyconnectedwith themethyl ester of loganic acid (loganin)Thebiological activity of loganic acid was recognised slightly and


849826

84789

1 854 829854

768

548 536548

698

856

71 682 718776 785

35455565758595

105

0 1 2 3 4 5Time (h)

ControlTimololStudy

IOP

(mm

Hg)

Figure 2 Mean values (mmHg) of IOP after intraconjunctivaladministration of the anthocyan-irdoid fraction (study) and timololThe data are expressed as the mean values and the error barsrepresent standard deviations Statistically significant values areshown in bold type

836 886803

914 889843812

725

551 576 536606

815

657

614

671 682 698

35455565758595

105115

0 1 2 3 4 5Time (h)

IOP

(mm

Hg)

ControlTimololStudy

Figure 3 Mean values (mmHg) of IOP after intraconjunctivaladministration of 07 loganic acid (study group) and timolol Thedata are expressed as the mean values and the error bars representstandard deviations Statistically significant values are shown in boldtype

its anti-inflammatory [47] antimicrobial [39] or antiprolifer-ative [48] properties were proven in the presence of the otheriridoids isolated from the corresponding plants

The effects of the anthocyan-irdoid fraction and pureloganic acid at a concentration of 07 on the IOP are pre-sented in Figures 2 and 3

The slight effect of the anthocyan-irdoid fraction wasinvestigated A 19 IOP decrease was measured 2-3 h afterocular administration This effect was weakened after 4 hDuring this assessment timolol gave 35 and 39 decreasesin IOP The last values are in agreement with the results ofother authors that observed similar IOP reduction in rat [49]or dog models [50]

More promising results were observed in the case ofocular administration of 07 loganic acid A clear statisti-cally significant (119901 lt 005) reduction in IOP 1 2 3 and 5hours after administration of 07 loganic acid was found

865798

768815

736

846810

754

659 689745

789779 815866 839 805

749

500550600650700750800850900950

0 1 2 3 4 5

IBF

(flux

)

Time (h)

ControlTimololStudy

Figure 4 Mean of iris blood flow (flux) after intraconjunctivaladministration of timolol and loganic acid (study) The data areexpressed as the mean values and the error bars represent standarddeviations Statistically significant values are shown in bold type

compared to the administration of the vehicle (Figures 2 and3) An approximately 25 statistically significant reductionin IOP was observed during the first 3 hours At the end ofthe experiment 5 hours after initiating the experiment a 15decrease was measured Although compared to the loganicacid timolol maleate gave a stronger effect on the IOP it isour opinion that this effect may indicate the possibility ofpractical use

One of several plant-origin compounds with proven IOP-reducing effect is forskolin obtained from Coleus forskohlii(Lamiaceae) Following intraconjunctival administration thecompound stimulates adenylate cyclase which leads to theactivation of cyclic adenosine monophosphate in ciliaryepithelium and a reduction in IOP resulting from the reducedproduction of aqueous fluid in rabbits monkeys and humans[13 14] Nowadays the only drug of natural origin currentlyused in glaucoma treatment is pilocarpine the alkaloid fromJaborandi (Pilocarpus microphyllus) leaves Its pharmacolog-ical activities are probably connected with its properties as amuscarinic agonist with no nicotinic effects [23 24]

The second cause of glaucoma is reduced bloodflow in theciliary arteries We measured the values of iris blood flow ofthe active compound that is loganic acidThe data are shownin Figure 4 The statistically significant (119901 lt 005) increase inthe mean iris blood flow was observed only 2 hours after theadministration of 07 loganic acid

No IOP-reducing effect was observed after oral adminis-tration of the Cornelian cherry fruit lyophilisate The evalu-ation of possible allergic reactions in the tested eyes was alsoperformed During the experiment the allergenic propertiesof the extract or loganic acid were evaluated using a visualmethod [51] No irritant or allergenic effect of the preparationat the administration time or during the experiment wasfound

Although there are great numbers of papers consideringthe effect of timolol as a hypotensive agent in eyes theexact mechanism is still unclear [52 53] The proposedmechanism of reduction of IOP consists of reducing thesecretion of aqueous humour in the ciliary body epitheliumby inhibiting 120573-adrenergic receptors According to other


authors [54] loganic acid isolated from Gentiana crassicaulispossesses anti-inflammatory properties by inhibitory effectson LPS-induced nitric oxide (NO) Also [55] shows that theproduction and increased concentration of NO in aqueoushumour are connected with the pathogenesis of glaucomaAn elevated level of NO was observed in primary open-angleglaucoma patients [56] We suspect that an NO-inhibitoryeffect of loganic acid could result in the observed IOP andthe blood flow in the eye

Very promising data obtained during the experimentcould be the first step towards clinical assays Althoughthey were achieved in an animal (rabbit) model accordingto our knowledge they could be related to human eyesfor glaucoma therapy Also this is the first ophthalmologyresearch in which the active compound belongs to a subclassof terpenoids that is iridoids Nowadays there are no dataabout the ocular pharmacological activity of other iridoidsso other compounds from this group should be taken intoconsideration

4 Conclusions

Intraconjunctival administration of loganic acid (07)reduces the IOP in rabbit eyes Our data obtained in ananimal model directly suggest that loganic acid could befurther used in the adjunct therapy of glaucoma in patientswith increased IOP It could also be beneficial for theintensification of vascular flows in diabetic and hypertensiveretinopathy or other conditions of ocular blood vessels (egvenous thrombosis or arterial embolism)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was realised with a grant for a scientific projectfor young scientists at the Wroclaw Medical University (no8Pbmn) a scientific scholarship within the framework ofthe project ldquoDevelopment Program of the Medical Academyin Wroclawrdquo realised with funds of the European SocialFund Operational ProgrammeHuman Capital (Contract noUDA-POKL040101-00-01008-01) and Programme of TheLeading National Research Centre (KNOW) for years 2014ndash2018 in Wroclaw Centre of Biotechnology

References

[1] D Ackland N Kumaran and R Zia ldquoTreating glaucoma thenot-so-nice guidancerdquoBritish Journal of Ophthalmology vol 98no 9 pp 1139ndash1140 2013

[2] I I Bussel and A A Aref ldquoDietary factors and the risk ofglaucoma a reviewrdquo Therapeutic Advances in Chronic Diseasevol 5 no 4 pp 188ndash194 2014

[3] D Peters B Bengtsson and A Heijl ldquoFactors associatedwith lifetime risk of open-angle glaucoma blindnessrdquo ActaOphthalmologica vol 92 no 5439 pp 421ndash425 2014

[4] M E Cavet J L Vittitow F Impagnatiello E Ongini and EBastia ldquoNitric oxide (NO) an emerging target for the treatmentof glaucomardquo Investigative Ophthalmology ampVisual Science vol55 no 8 pp 5005ndash5015 2014

[5] D J Rhee ldquoPreventing glaucoma in a high-risk populationimpact and observations of the ocular hypertension treatmentstudyrdquo Archives of Ophthalmology vol 127 no 2 pp 216ndash2182009

[6] L J Rosenbaum ldquoManagement of patients with ocular hyper-tension a cost-effectiveness approach from the ocular hyper-tension treatment studyrdquoThe American Journal of Ophthalmol-ogy vol 143 no 1 pp 191ndash192 2007

[7] M Mulak D Szumny A Sieja-Bujewska and M KubrakldquoHeidelberg edge perimeter employment in glaucoma diagno-sis preliminary reportrdquo Advances in Clinical and ExperimentalMedicine vol 21 no 5 pp 665ndash670 2012

[8] F Aptel A Begle A Razavi et al ldquoShort- and long-term effectson the ciliary body and the aqueous outflow pathways of high-intensity focused ultrasound cyclocoagulationrdquo Ultrasound inMedicine amp Biology vol 40 no 9 pp 2096ndash2106 2014

[9] R A Coulter J D Bartlett and R G Fiscella ldquoPharmacother-apy of the ophthalmic patientrdquo inClinical Ocular PharmacologyJ D Bartlett and S D Jaanus Eds chapter 1 pp 3ndash15Butterworth-Heinemann St LouisMoUSA 5th edition 2008

[10] H D Jampel ldquoPharmacotherapy in Glaucoma edited by SelimOrgul and Josef Flammer Bern Verlag Hans Huber 2000 333pages illusrdquo Survey of Ophthalmology vol 46 no 2 p 192 2001

[11] T V Johnson and S I Tomarev ldquoRodent models of glaucomardquoBrain Research Bulletin vol 81 no 2-3 pp 349ndash358 2010

[12] S I Tomarev ldquoAnimal models of glaucomardquo in Encyclopedia ofthe Eye D A Dartt Ed pp 106ndash111 Academic Press OxfordUK 2010

[13] J Caprioli and M Sears ldquoCombined effect of forskolin andacetazolamide on intraocular pressure and aqueous flow inrabbit eyesrdquo Experimental Eye Research vol 39 no 1 pp 47ndash501984

[14] V Gunasekera E Ernst and D G Ezra ldquoSystematic Internet-based review of complementary and alternative medicine forglaucomardquo Ophthalmology vol 115 no 3 pp 435e2ndash439e22008

[15] H T Balaydn M Senturk S Goksu and A Menzek ldquoSyn-thesis and carbonic anhydrase inhibitory properties of novelbromophenols and their derivatives including natural productsVidalol Brdquo European Journal ofMedicinal Chemistry vol 54 pp423ndash428 2012

[16] J C Merritt W J Crawford P C Alexander A L Anduze andS S Gelbart ldquoEffect of marihuana on intraocular and bloodpressure in glaucomardquo Ophthalmology vol 87 no 3 pp 222ndash228 1980

[17] S Pinar-Sueiro R Rodrıguez-Puertas and E Vecino ldquoCanna-binoid applications in glaucomardquo Archivos de la SociedadEspanola de Oftalmologia vol 86 no 1 pp 16ndash23 2011

[18] P Trittibach B E Frueh and D Goldblum ldquoBilateral angle-closure glaucoma after combined consumption of lsquoecstasyrsquo andmarijuanardquo The American Journal of Emergency Medicine vol23 no 6 pp 813ndash814 2005

[19] I Ilieva K Ohgami K Shiratori et al ldquoThe effects of Ginkgobiloba extract on lipopolysaccharide-induced inflammation invitro and in vivordquo Experimental Eye Research vol 79 no 2 pp181ndash187 2004


[20] X-S Mi J-X Zhong R C-C Chang and K-F So ldquoResearchadvances on the usage of traditional Chinese medicine forneuroprotection in glaucomardquo Journal of Chinese IntegrativeMedicine vol 11 no 4 pp 233ndash240 2013

[21] L Quaranta S Bettelli M G Uva F Semeraro R Turano andE Gandolfo ldquoEffect of Ginkgo biloba extract on preexistingvisual field damage in normal tension glaucomardquo Ophthalmol-ogy vol 110 no 2 pp 359ndash362 2003

[22] S Dorairaj R Ritch and J M Liebmann ldquoVisual improvementin a patient taking Ginkgo Biloba extract a case studyrdquo ExploreThe Journal of Science and Healing vol 3 no 4 pp 391ndash3952007

[23] L A Dethloff T Chang and C L Courtney ldquoToxicologicalcomparison of a muscarinic agonist given to rats by gavage orin the dietrdquo Food and Chemical Toxicology vol 34 no 4 pp407ndash422 1996

[24] N Z Gharagozloo S J Relf and R F Brubaker ldquoAqueousflow is reduced by the alpha-adrenergic agonist apraclonidinehydrochloride (ALO 2145)rdquo Ophthalmology vol 95 no 9 pp1217ndash1220 1988

[25] A McIntyre ldquoThe eyesrdquo in Herbal Treatment of ChildrenA McIntyre Ed chapter 13 pp 285ndash291 Butterworth-Heinemann Edinburgh Scotland 2005

[26] B Saric-Kundalic C Dobes V Klatte-Asselmeyer and J SaukelldquoEthnobotanical study on medicinal use of wild and cultivatedplants in middle south and west Bosnia and HerzegovinardquoJournal of Ethnopharmacology vol 131 no 1 pp 33ndash55 2010

[27] RMokkapatti ldquoAn experimental double-blind study to evaluatethe use of Euphrasia in preventing conjunctivitisrdquo BritishHomoeopathic Journal vol 81 no 1 pp 22ndash24 1992

[28] M Loo ldquoConjunctivitisrdquo in Integrative Medicine for ChildrenM Loo Ed chapter 25 pp 263ndash268 WB Saunders St LouisMo USA 2009

[29] T Sozanski A Kucharska A Szumny et al ldquoThe protectiveeffect of the Cornus mas fruits (cornelian cherry) on hyper-triglyceridemia and atherosclerosis through PPAR120572 activationin hypercholesterolemic rabbitsrdquo Phytomedicine vol 21 no 13pp 1774ndash1784 2014

[30] A Z Kucharska A Szumny and JOszmianski ldquoMethod of pre-paring of loganic acidrdquo Polish Patent no 388632 2010 (Polish)

[31] M Leiva C Naranjo and M T Pena ldquoComparison of therebound tonometer (ICare) to the applanation tonometer(Tonopen XL) in normotensive dogsrdquo Veterinary Ophthalmol-ogy vol 9 no 1 pp 17ndash21 2006

[32] S Prabhakar B Mahesh and M Shanthamallappa ldquoA com-parative study of intraocular pressure measurement by threetonometers in normal subjectsrdquo Nepalese Journal of Ophthal-mology vol 5 no 2 pp 201ndash206 2013

[33] S Salim H Du and J Wan ldquoComparison of intraocularpressure measurements and assessment of intraobserver andinterobserver reproducibility with the portable icare reboundtonometer and goldmann applanation tonometer in glaucomapatientsrdquo Journal of Glaucoma vol 22 no 4 pp 325ndash329 2013

[34] C Schweier J V M Hanson J Funk and M Toteberg-HarmsldquoRepeatability of intraocular pressure measurements with IcarePRO rebound Tono-Pen AVIA and Goldmann tonometers insitting and reclining positionsrdquo BMC Ophthalmology vol 13article 44 2013

[35] AM Pawlowska F Camangi andA Braca ldquoQuali-quantitativeanalysis of flavonoids ofCornusmasL (Cornaceae) fruitsrdquo FoodChemistry vol 119 no 3 pp 1257ndash1261 2010

[36] K Chan ldquoThe Pharmacology of Chinese Herbs Kee ChangHuang Published 1993 CRC Press Inc Boca Raton FL 388pages ISBN 0 8493 4915 X m10000rdquo Journal of Pharmacy andPharmacology vol 46 no 2 pp 159ndash160 1994

[37] W Ma K J Wang C-S Cheng et al ldquoBioactive compoundsfrom Cornus officinalis fruits and their effects on diabeticnephropathyrdquo Journal of Ethnopharmacology vol 153 no 3 pp840ndash845 2014

[38] S Quideau ldquoFlavonoids Chemistry Biochemistry and Appli-cations Edited by Oslashyvind M Andersen and Kenneth RMarkhamrdquo Angewandte Chemie International Edition vol 45no 41 pp 6786ndash6678 2006

[39] M Wu P Wu M Liu H Xie Y Jiang and X Wei ldquoIridoidsfromGentiana loureiriirdquo Phytochemistry vol 70 no 6 pp 746ndash750 2009

[40] X ZhangQXuHXiao andX Liang ldquoIridoid glucosides fromStrychnos nux-vomicardquo Phytochemistry vol 64 no 8 pp 1341ndash1344 2003

[41] S Deng B J West and C J Jensen ldquoUPLC-TOF-MS char-acterization and identification of bioactive iridoids in cornusmas fruitrdquo Journal of Analytical Methods in Chemistry vol 2013Article ID 710972 7 pages 2013

[42] C Bergeron AMarston R Gauthier andKHostettmann ldquoIri-doids and secoiridoids fromGentiana linearisrdquo Phytochemistryvol 44 no 4 pp 633ndash637 1997

[43] J S Kim ldquoSeeds of Cornus officinalis and diabetic cataractsrdquoin Handbook of Nutrition Diet and the Eye V R Preedy Edchapter 45 pp 451ndash458 Academic Press SanDiego Calif USA2014

[44] W Ma K-J Wang C-S Cheng et al ldquoBioactive compoundsfrom Cornus officinalis fruits and their effects on diabeticnephropathyrdquo Journal of Ethnopharmacology vol 153 no 3 pp840ndash845 2014

[45] W-L Jiang S-P Zhang J Hou and H-B Zhu ldquoEffect ofloganin on experimental diabetic nephropathyrdquo Phytomedicinevol 19 no 3-4 pp 217ndash222 2012

[46] P-C Hsu Y Tsai J Lai C Wu S Lin and C Huang ldquoInte-grating traditional Chinese medicine healthcare into diabetescare by reducing the risk of developing kidney failure amongtype 2 diabetic patients a population-based case control studyrdquoJournal of Ethnopharmacology vol 156 pp 358ndash364 2014

[47] M Del Carmen Recio R M Giner S Manez and J L RiosldquoStructural considerations on the iridoids as anti-inflammatoryagentsrdquo Planta Medica vol 60 no 3 pp 232ndash234 1994

[48] Z L ZhouW Yin H Zhang Z Feng and J Xia ldquoA new iridoidglycoside and potential MRB inhibitory activity of isolatedcompounds from the rhizomes of Cyperus rotundus LrdquoNaturalProduct Research vol 27 no 19 pp 1732ndash1736 2013

[49] M Seki T Tanaka H Matsuda et al ldquoTopically adminis-tered timolol and dorzolamide reduce intraocular pressure andprotect retinal ganglion cells in a rat experimental glaucomamodelrdquoBritish Journal of Ophthalmology vol 89 no 4 pp 504ndash507 2005

[50] C E Plummer E O MacKay and K N Gelatt ldquoComparisonof the effects of topical administration of a fixed combinationof dorzolamide-timolol to monotherapy with timolol or dorzo-lamide on IOP pupil size and heart rate in glaucomatous dogsrdquoVeterinary Ophthalmology vol 9 no 4 pp 245ndash249 2006

[51] C Baudouin ldquoAllergic reaction to topical eyedropsrdquo CurrentOpinion in Allergy and Clinical Immunology vol 5 no 5 pp459ndash463 2005


[52] L C Ho I P Conner C Do et al ldquoIn vivo assessment ofaqueous humor dynamics upon chronic ocular hypertensionand hypotensive drug treatment using gadolinium-enhancedMRIrdquo Investigative Ophthalmology amp Visual Science vol 55 no6 pp 3747ndash3757 2014

[53] A H Neufeld S P Bartels and J H K Liu ldquoLaboratory andclinical studies on the mechanism of action of timololrdquo Surveyof Ophthalmology vol 28 supplement 1 pp 286ndash290 1983

[54] T Lv M Xu D Wang et al ldquoThe chemical constituents fromthe roots of Gentiana crassicaulis and their inhibitory effects oninflammatorymediators NO and TNF-120572rdquoNatural Products andBioprospecting vol 2 no 5 pp 217ndash221 2012

[55] S-H Chiou C-J Chang W-M Hsu et al ldquoElevated nitricoxide level in aqueous humor of patients with acute angle-closure glaucomardquoOphthalmologica vol 215 no 2 pp 113ndash1162001

[56] F Galassi A Sodi F Ucci G Renieri B Pieri and E MasinildquoOcular haemodynamics and nitric oxide in normal pressureglaucomardquo Acta Ophthalmologica Scandinavica Supplementvol 78 no 232 pp 37ndash38 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Only a few studies have been aimed at the effect ofcompounds of natural origin on the IOP as well as otherglaucoma-progressing factors such as vascular blood flowantioxidative effects or neuroprotection The majority ofthese studies were conducted in vitro but a few were invivo and reached clinical trials The difficulties encounteredin the evaluation of natural compounds on the progress ofglaucoma-associated neuropathy are a consequence of long-term disease development but are also because of the limitedpossibility to use proper animal models [11 12]

One of several plant-origin compounds with a provenIOP-reducing effect is forskolin which is obtained fromColeus forskohlii (Lamiaceae) Following intraconjunctivaladministration the compound stimulates adenylate cyclasewhich leads to the activation of cyclic adenosine monophos-phate in ciliary epithelium and the reduction of IOP resultingfrom reduced production of aqueous products in rabbitsmonkeys and humans [13 14] Similarly natural bromophe-nols isolated from red algae belonging to the Rhodomelaceaefamily such as vidalol B present activity as carbonic anhy-drase inhibitors and may become a valuable IOP-reducingdrug in the future [15] Recently marijuana was consideredas an IOP-reducing natural compoundHowever because of ahigh ratio of nonresponders short half-life and considerabletoxicity the substance was not qualified for medicinal use[16ndash18] Among other plants Ginkgo biloba leaf extracts maybe used for adjunct glaucoma therapy The extract containspolyphenolic flavonoid compounds as well as terpenoidsand is able to scavenge free radicals at themitochondrial levelin order to improve blood flow In addition the substanceprevents neurotoxicity of glutamate whichmay be importantfor a neuroprotective effect and inhibits lipid peroxidation[19 20] Although it does not affect the IOP it may improvevascular blood flow into the optic nerve [21] Clinical trialshave indicated that anthocyanins and Ginkgo biloba extractmay be effective in improving the visual function and thevisual field of patients with normal-tension glaucoma [2022] Nowadays the only drug of natural origin currentlyused in glaucoma treatment is pilocarpine which is thealkaloid from Jaborandi (Pilocarpus microphyllus) leaves Itspharmacological activities are connected with properties ofa muscarinic agonist with no nicotinic effects [23 24] Oneof the most popular plant extracts traditionally used in eyesdiseases in Europe especially for the inflammation of eyesis Eyebright (Euphrasia) Its most important constituentsthat affect the eyes are iridoid glycosides including aucubingeniposide catalpol and luproside [25ndash27] Although thereare no relevant clinical studies an established traditional useof those eye drops proves their efficacy in conjunctivitis [28]

To the best of our knowledge there is a lack of dataregarding the effect of loganic acid or Cornelian cherrypreparations on IOP or vascular flow in the eye We havenot found any loganic-acid-containing or Cornelian-cherry-based preparations registered in the world which couldbe used for the therapy of glaucoma reduction of IOP orincreasing vascular blood flow in the eye

The aim of our study was to investigate the influence ofCornus mas iridoid-rich extract as well as its predominantcompound loganic acid on IOP

2 Materials and Methods

21 Evaluation of Cornelian Cherry Constituents Raciborski-variety Cornelian cherries (Cornus mas L) were obtainedfrom the Bolestraszyce Arboretum and Institute of Phys-iography Poland in September 2012 The plant materialwas authenticated by Professor Jakub Dolatowski and thevoucher specimen (12402) was deposited at the Herbariumof Arboretum and Institute of Physiography in BolestraszycePoland The polyphenolic and loganic acid iridoid fractionswere prepared in the Department of Fruit Vegetable andCereals Technology at Wroclaw University of Environmentaland Life Science according to the procedure described inour previous publication [29] and patent [29] Ripe Cor-nelian cherries were washed frozen and stored at minus20∘Cuntil processing After freezing removing stones defrostingand depectinisation we obtained the juice The juice waspurified on an Amberlite XAD-16 resin column Iridoidsand phenolics were eluted with 80 ethanol The eluent wasconcentrated under vacuum at 40∘C and lyophilised About20 g of lyophilisate from the iridoid-polyphenolic fractionwas obtained from 5 kg of fruit After freeze drying thesamples were ground into a powder using a laboratory millThe structure of the compounds in the iridoid-phenolicfraction was determined by UPLC-MS analysis

22 Isolation of Anthocyan-Irdoid Fraction and Pure LoganicAcid The easy effective and low-cost approach for isolationof loganic acid as well as the anthocyanin fraction wasdeveloped in our research group [30] In brief freeze-diedCornelian fruit powder (as described above) was dissolvedin 30 ethanol and separated using column chromatography(polyamide) Elution with 50 ethanol (and 1 acetic acid)afforded the anthocyanin fraction and pure loganic acidApplying the described procedure to 1 kg of fresh fruitallowed us to obtain 40 g of the anthocyanin fraction and15 g of pure loganic acid

The structure and purity of loganic acid was determinedby NMR and UPLC-MS analyses

23 Identification of Compounds by UPLC-MSMS andHPLCCompounds of the phenolic-iridoid and loganic acid frac-tions were identified by applying the method described byKucharska et al [30] using the acuity ultra-performanceliquid chromatography (UPLC) system coupled with aquadrupole time-of-flight (Q-TOF) MS instrument (WatersCorp Milford MA USA) with an electrospray ionisation(ESI) source Separation was achieved on the Acquity BEHC18 column (100mm times 21mm id 17mm Waters) Detec-tion wavelengths were set to 245 and 520 nm The mobilephase was a mixture of 45 formic acid (A) and acetonitrile(B) The gradient program was as follows initial conditions99 (A) 12min 75 (A) 125min 100 (B) 135min 99(A)The flow rate was 045mLmin and the injection volumewas 5 120583L The column was operated at 30∘C The instrumentwas operated in both positive- (anthocyanins) and negative-ion (iridoids) modes scanning the mz from 100 to 1500 ata scan rate of 20 scycle The major operating parameters for



























6) 120575 (ppm) 16856 -CO

2H 15055 C-3 11305 C-4


6) 120575 (ppm) 729




O

O

OOH

OH

OH

OH

OH

O

HO

H

HH3C




849826

84789

1 854 829854

768

548 536548

698

856

71 682 718776 785

35455565758595

105

0 1 2 3 4 5Time (h)

ControlTimololStudy

IOP

(mm

Hg)


836 886803

914 889843812

725

551 576 536606

815

657

614

671 682 698

35455565758595

105115

0 1 2 3 4 5Time (h)

IOP

(mm

Hg)

ControlTimololStudy






865798

768815

736

846810

754

659 689745

789779 815866 839 805

749

500550600650700750800850900950

0 1 2 3 4 5

IBF

(flux

)

Time (h)

ControlTimololStudy










4 Conclusions




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































6) 120575 (ppm) 16856 -CO

2H 15055 C-3 11305 C-4


6) 120575 (ppm) 729




O

O

OOH

OH

OH

OH

OH

O

HO

H

HH3C




849826

84789

1 854 829854

768

548 536548

698

856

71 682 718776 785

35455565758595

105

0 1 2 3 4 5Time (h)

ControlTimololStudy

IOP

(mm

Hg)


836 886803

914 889843812

725

551 576 536606

815

657

614

671 682 698

35455565758595

105115

0 1 2 3 4 5Time (h)

IOP

(mm

Hg)

ControlTimololStudy






865798

768815

736

846810

754

659 689745

789779 815866 839 805

749

500550600650700750800850900950

0 1 2 3 4 5

IBF

(flux

)

Time (h)

ControlTimololStudy










4 Conclusions




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















849826

84789

1 854 829854

768

548 536548

698

856

71 682 718776 785

35455565758595

105

0 1 2 3 4 5Time (h)

ControlTimololStudy

IOP

(mm

Hg)


836 886803

914 889843812

725

551 576 536606

815

657

614

671 682 698

35455565758595

105115

0 1 2 3 4 5Time (h)

IOP

(mm

Hg)

ControlTimololStudy






865798

768815

736

846810

754

659 689745

789779 815866 839 805

749

500550600650700750800850900950

0 1 2 3 4 5

IBF

(flux

)

Time (h)

ControlTimololStudy










4 Conclusions




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















4 Conclusions




Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article application of cornelian cherry iridoid...

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