Research ArticleAssessment of Lemon Balm (Melissa officinalis L) HydrogelsQuality and Bioactivity in Skin Cells

Kristina Ramanauskien1 Ada Stelmakiene1 and Daiva Majien23

1Department of Clinical Pharmacy Lithuanian University of Health Sciences A Mickeviciaus Street 9 Kaunas Lithuania2Department of Drug Technology and Social Pharmacy Lithuanian University of Health SciencesA Mickeviciaus Street 9 Kaunas Lithuania3Neuroscience Institute Lithuanian University of Health Sciences EiveniJ Street 4 Kaunas Lithuania

Correspondence should be addressed to Ada Stelmakiene adastelmakieneyahoocom

Received 22 July 2015 Revised 22 September 2015 Accepted 7 October 2015

Academic Editor Yuri Clement

Copyright copy 2015 Kristina Ramanauskiene et alThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

The aim of the study was to design gels with lemon balm extract assess their quality and investigate the effect of rosmarinic acidon skin cells in normal conditions and under oxidative stressMethods The quantities of rosmarinic acid (RA) released from gelswere evaluated by applying the HPLC technique HaCaT cell viability was assessed by using the MTTmethod ROS generation wasmeasured using DCFH-DA dye The results showed that the gelling material affected the release of RA content from gels Lowerand slower RA content release was determined in carbomer-based gels After 6 hours of biopharmaceutical research in vitro atleast 4 of RA was released from the gel The results of the biological studies on HaCaT cells demonstrated that in the oxidativestress conditions RA reduced intracellular ROS amounts to 28 025ndash05mgmL of RA increased cell viability by 10ndash24 andprotected cells from the damage caused by H

2O2 Conclusions According to research results it is appropriate to use a carbomer as

the main gelling material and its concentration should not exceed 10 RA depending on the concentration reduces the amountof intracellular ROS and enhances cell viability in human keratinocytes in oxidative stress conditions

1 Introduction

Lemon balm (Melissa officinalis L) and its preparations havea mildly soothing antiviral effect improve the digestivetract and relax intestinal spasms [1 2] Lemon balm is anatural source of rosmarinic acid (RA) RA is one of themain phenolic acids in the chemical composition of Melissaofficinalis L and it determines the pharmacological effectand the medical use of the plant [2ndash5] Current studies haveshown that lemon balm preparations have bacteriostaticantimicrobial and antiviral effects [3] Currently there arevery comprehensive surveys that attempt to show the effect oflemon balm against herpes simplex virus [6ndash10] Since lemonbalm has antiviral effects some authors state that they can beused against HIV-1 infection In vitro studies have shown thatlemon balm preparations inhibit HIV-1 reverse transcriptase[11] There is evidence in scientific literature that lemon balmhas antihistamine effects thus it can be used externally by

placing the cut grass on insect bites or other irritated areas[6 12] RA in lemon balm has demonstrated more activeantioxidant activity compared to 120572-tocopherol [13] Due toits antioxidant anti-inflammatory and immunomodulatoryeffects [3] RA as part of the chemical composition of lemonbalm is effective in relieving symptoms of atopic dermatitisThe in vivo study conducted by Lee et al (2008) showedthat people with atopic dermatitis who applied RA emulsiondaily had a decrease of erythema after 4ndash8 weeks and hadthe skin water loss decreased after 8 weeks [2] It can beargued that because of the antimicrobial antiviral anti-inflammatory and antioxidant properties Melissa officinalisL can be used as a natural source of RA in the productionof semisolid preparations characterized by antimicrobial andprotective effects

When modeling a semisolid preparation it is highlyimportant to choose appropriate base substances becausethey determine the physicochemical properties and

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 635975 7 pageshttpdxdoiorg1011552015635975

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Composition of experimental lemon balm hydrogels

Composition () GelsN1 N2 N3 N4 N5 N6 N7 N8 N9

Carbomer 980 05 10 15 05 05 05 mdash mdash mdashMethylcellulose 15cP mdash mdash mdash 10 20 40 10 20 40Propylene glycol 200 200 200 200 200 200 200 200 20010 NaOH qs ad pH 7 qs ad pH 7 qs ad pH 7 qs ad pH 7 qs ad pH 7 qs ad pH 7 mdash mdash mdashMelissa officinalis (L)dry extract 40 40 40 40 40 40 40 40 40

Purified qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000

therapeutic effects of the final product [14] A suitablebase ensures the stability of semisolid preparations duringstorage good distribution on the skin and an efficient releaseof a drug substance from the preparation [14] Creams andhydrophilic gels are the most common topical preparationsapplied on the skin due to their positive sensory propertiesThe advantage that gel has over ointments or cream is that itis a more stable nonslimy and semisolid form of medication[15] Due to its characteristic sensory properties (easilyapplicable on the skin easily cleaned with water and leavingno greasy membrane on skin unlike other ointments) gelis suitable for the modeling of natural preparations thatare usually applied several times a day for longer periodsof time Gel ensures easy and safe administration of suchpreparations at home by nonmedical persons [16] Becauseof the aforementioned properties it is important to producegel containing lemon balm extract and to assess its qualityusing biopharmaceutical tests Quality control testing ofsuch topical semisolid dosage forms includes the identityquantitative analysis homogeneity rheological propertiesparticle size determination consistency and medicinalsubstance release tests (Eur Ph 60 0120080132) Invitro release testing identifies the influence of a number ofphysicochemical parameters such as solubility of the drugsubstance in the carrier or the effect of particle size andviscosity of the dosage form on the release of the medicinalsubstance from the dosage form through a syntheticmembrane into the acceptor medium [17 18] The resultsof scientific research revealed that rosmarinic acid is moreeasily released from emulsion bases than from ointments[19] Because of the lack of data of biopharmaceuticalscientific tests it is important to assess the rate of RA releasefrom modulated hydrophilic gels

The aim of the study was to design pharmaceuticalhydrophilic semisolid dosage forms with lemon balm extractassess their quality and investigate the protective effect ofthe main active substance (rosmarinic acid) on skin cells innormal conditions and under oxidative stress

2 Materials and Methods

Experimental lemon balm hydrogel formulations were pre-pared according to the general technological principles ofsemisolid preparations [20] The compositions of the formu-lated gels are presented in Table 1 These gels were developed

to study the effect of individual gelling polymers on drugrelease behavior Dry lemon balm extract was dissolved inwater and dispersed in swelled gels under stirring Carbomergels (N1ndashN3) were prepared by neutralization with sodiumhydroxide Gels of methylcellulose (N7ndashN9) were preparedby overnight soaking of the polymer for complete hydrationCombination gels (N4ndashN6) were prepared by mixing poly-mer gels together [21]

21 Physicochemical and Biopharmaceutical Properties of GelsThe dynamic viscosity of gels was determined at room tem-perature using a Vibro Viscometer SV-10 (A amp D CompanyLtd Japan) The studied substance was placed into a specialcontainer for measurement Subsequently the container wasfixed on the working surface of the device and sensors weresubmerged into the studied substance The rotation speed ofa cylindrical spindle was 100 revmin The length of everymeasurement was 10 sec (119899 = 3)

The pH of semisolid preparations was analyzed using apH meter HD 21051 (Delta OHM Italy) A 5 solution wasprepared to determine pH levels The appropriate amount ofthe semisolid formula was topped with purified water andstirred for 30minutes on an IKAMAGC-MAGHS7magneticstirrer (IKA-Werke GmbH amp Co KG Staufen Germany) ata temperature of 50∘C Then the solution was cooled andfiltered through a paper filter

In vitro release experiments were performed using mod-ified Franz-type diffusion cells The semisolid sample (100 plusmn002 g) was placed into the cell with a dialysis membraneThe dialysis membrane Cuprophan (Medicell InternationalLtd UK) was made of natural cellulose The area of thediffusion was 177 cm2 Purified water acted as the acceptormedium The temperature of the acceptor medium was keptat 37plusmn02∘CThemediumwas stirred using amagnetic stirrerSamples from the acceptor solution were taken at 1 2 4 and6 h and were immediately replaced with the same volume offresh acceptor solution

Lemon balm extracts were analyzed by applying high-performance liquid chromatography The capillary HPLCmethod was developed and validated The amount of theRA was evaluated by applying the validated HPLC methodusing the Agilent 1260 Infinity Capillary LC System (AgilentTechnologies USA) with an Agilent Diode Array Detec-tor The separation was performed in a C18 ACE (5120583m)150 times 05 id column The mobile phase consisted of solvents

Evidence-Based Complementary and Alternative Medicine 3

A (05 aqueous solution of acetic acid 119881119881) and B (ace-tonitrile) using the following gradient elution 23 of Bat 0min 40 of B at 10min and 70 of B at 11ndash15minit subsequently returned to the initial conditions with 10-minute reequilibration with the total run time of 25 minutesThe analysis was carried out at a flow rate of 10 120583Lminminus1 withthe detection wavelength set at 330 nm

22 Cell Lines and Cell Culture Human keratinocyte cell lineHaCaT was purchased from the Cell Lines Service GmbH(Germany) Cells of convenient concentration were seededin culture flasks containing DMEM with 10 of fetal bovineserum 100UmL penicillin and 100 120583gmL streptomycinCultures were then incubated at 37∘C with 5 CO

2and

saturated humidity culture transfer was performed once aweek and the medium was renewed twice a week

23 Measurement of Cell Viability Twenty-four hours priorto testing cells with the investigated preparations were trans-ferred to 96-well plates at concentrations of 30000 cellswellThe cells were incubated with (A) different concentrationsof H2O2and (B) different concentrations of RA and H

2O2

After 24-hour treatment with the preparations the DMEMmedium was removed and the cells were washed twice with100mLwell warm Phosphate Buffered Saline (PBS) Afterwashing 180 120583Lwell PBS was added along with 20120583Lwellof 5mgmLMTT dye dissolved in PBS to each well The cellswere incubatedwithMTT at 37∘C for 2 hours Afterwards thedyewas removed the intracellularly formed crystals were dis-solved in DMSO (100 120583Lwell) and the plate was kept in thedark for 15 minutes The absorption was measured at 570 nmand 620 nm as reference with a microplate spectrophotome-ter (Sunrise Tecan Group Ltd Switzerland)The results wereexpressed as a fluorescence percentage in control cells

24 Measurement of Intracellular ROS Generation The pro-duction of ROS was assessed using the 2101584071015840-dichlorofluores-cein diacetate (DCFH-DA) as described in [22] Twenty-fourhours prior to treating cells with RA they were transferredto 96-well plates at concentrations of 50000 cellswell After24 hours the medium was removed and cells were washedwith PBS After washing 200120583Lwell HBSS medium sup-plemented with DCFH-DA (10 120583M) was added The cellswere incubated at 37∘C for 30min After the incubation thecells were washed twice with PBS and were subsequentlytreated with (A) different concentrations (005ndash05mg) ofRA or (B) 100120583M of H

2O2and different concentrations of

RA The fluorescence of DCF was detected by a fluorometerat excitation and emission of 488 and 525 nm wavelengthsrespectively Data are presented as means of fluorescenceintensity plusmn SE

25 Statistical Analysis of the Results All tests were donein triplicate Mean values and standard deviations of theresults were calculated by using Microsoft Office Excel 2010and SigmaPlot version 120 (Systat Software Inc) softwareThe significance of differences in test results was assessed byusing Studentrsquos 119905-test The differences were considered to bestatistically significant when 119901 lt 005

3 Results and Discussion

31 Evaluation of Physicochemical Properties ofHydrogels Forsemisolid preparations that could be applied to the treat-ment of atopic dermatitis natural medicinal plant materialdry lemon balm extract containing 80mgg of rosmarinicacid was selected as the active substance Gels producedwith lemon balm extract were homogeneous and yellowishto brownish in color According to the results (Table 2)gels that were produced with carbomer and the mixtureof carbomer and methylcellulose [N1ndashN6] had higher pHvalues Meanwhile gels produced with the gelling materialof methylcellulose [N7ndashN9] had weakly acidic pH the valuesranged from 584 to 632 It was observed that when theconcentration of methylcellulose in these gels increased thepH value of the gels was also increasing and they became lessacidic

The results in Table 2 show that the selected gellingmaterial affected the viscosity of the formulated gels Gels[N1ndashN6] in which carbomer was used as the main gellingmaterial had a higher viscosity whereas methylcellulose-based gels [N7ndashN9] had the lowest viscosity The lattersemisolid systems were characterized by a liquid consistencyThis shows that carbomer is a stronger gelling materialcompared to methylcellulose The results confirmed the datapublished in scientific literature indicating that the concen-tration of gelling material has an influence on the viscosityof semisolid preparations that is increasing carbomer andmethylcellulose concentrations in semisolid preparationsincreases their dynamic viscosity [15 23] Gels [N1ndashN3] inwhich carbomer was used as the main gelling material hadthe highest viscosity whereas the lowest dynamic viscositywas found in methylcellulose-based gels [N7ndashN9] Based onscientific literature the pH value of dermatological prepara-tions is an important quality indicator In order to reach themaximum effect of these preparations their pH should beclose to that of the skin (54 to 59) [24] In patients withatopic dermatitis the pH of the skin may increase (60 to65) [25] The results of our study showed that the producedsemisolid preparations are suitable for use on the skin theyare homogeneous and have suitable pH values and acceptableorganoleptic properties

32 Release of Rosmarinic Acid from Hydrogels The resultsshowed that the selected gelling material affected the releaseof RA content from semisolid systems (Figure 1) Lower andslower RA content release was more observed in carbomerand carbomer-methylcellulose-based combination gels thanin the methylcellulose gels alone In the evaluation of gelscontaining carbomer [N1ndashN3] as the main gelling materialthe highest RA content was released after 2 hours afterwhich time a slowdown of the release was observed SlowerRA content release kinetics was observed in carbomer-methylcellulose-based gels [N4ndashN6]The highest RA contentwas released after 4 hours and then the release sloweddown The highest RA content was released from gels inwhich methylcellulose was used as the gelling material Dataof the statistical analysis showed a statistically significantdifference (119901 lt 005) between RA quantities released from

4 Evidence-Based Complementary and Alternative Medicine

Table 2 Results of quality control testing in gels

Quality parameter GelsN1 N2 N3 N4 N5 N6 N7 N8 N9

pH value 745 plusmn 022 762 plusmn 031 775 plusmn 018 735 plusmn 021 720 plusmn 019 708 plusmn 013 584 plusmn 015 614 plusmn 027 632 plusmn 030Dynamic viscosity Pa s 245 plusmn 012 351 plusmn 008 386 plusmn 010 264 plusmn 014 290 plusmn 008 298 plusmn 003 001 plusmn 001 007 plusmn 001 061 plusmn 008

02468

101214161820

N1 N2 N3 N4 N5 N6 N7 N8 N9Samples

Rosm

arin

ic ac

id co

nten

t (

)

1h2h

4h6h

Figure 1 The kinetics of rosmarinic acid release from gels

methylcellulose-based hydrogels Even though the highestreleased RA content was found inmethylcellulose-based gelscarbomer due to its semisolid consistency is a more appro-priate base for the insertion of liquid lemon balm extractsMeanwhile methylcellulose can be used for the productionof gels in order to achieve a prolonged action of themedicinalpreparation Biopharmaceutical research proved that whenthe concentration of gelling materials in semisolid dosageforms is increasing the released RA content is decreasingIn vitro study of released RA is an informative tool for theassessment of the suitability of the base as a carrier for theinsertion of liquid lemon balm extract Scientific studieshave shown that RA was accumulating in the epidermis andthe hydrophilic dermis acted as a barrier preventing deeperpenetration and entry of RA into systemic blood flow [19]Substance penetration into or through the skin occurs in acoherent sequence when dissolved molecules released fromthe dosage form reach the surface of the stratumcorneumandpenetrate through it [26] Materials can penetrate throughthe skin by intracellular extracellular and additional ldquoshuntrdquo(through hair follicles and gland ducts) routes [27 28]Substance penetration using the intracellular polar routeoccurswhenmolecules diffuse through the cytoplasmof deadkeratinocytes and the surrounding lipid matrix [29] For thisreason the human keratinocyte cell line HaCaT was chosenfor further studies of the biological effects of the releasedRA With these studies we aimed at determining whetherthe RA released from the gel has protective effect in normalconditions and under oxidative stress

33 Influence of H2O2 and Rosmarinic Acid on HaCaTCell Viability during H2O2 Exposure In order to assess theeffect of different concentrations of H

2O2on HaCaT cell

viability the MTT assay was carried out The results of theseexperiments are presented in Figure 2(a) EC

50was estimated

to be 1837 plusmn 204 120583MH2O2

For further experiments we chose RA concentrations(005ndash05mgmL) according to the results of RA release fromthe gel received at various time periods and incubated thecells for 24 hours The results of our experiments revealed(Figure 2(b)) that higher concentrations of RA (025ndash05mgmL) protected the cells from the damage caused bydifferent concentrations of H

2O2and enhanced cell viability

by 10ndash24 Moreover 05mgmL of RA restored HaCaT cellviability until the control level in the presence of 100120583M ofH2O2

It is possible that the protective effect of RA is relatedto its antioxidant activity Oxidative stress conditions in cellsdevelop in the presence of an imbalance between ROS gen-eration and endogenous antioxidant defense mechanismsThis state of oxidative stress can affect all important cellularcomponents like proteins DNA andmembrane lipids whichis considered to be the main mechanism of cell damage[30] There is research confirming that exogenous moleculesfrom dietary sources such as polyphenols are very efficientin preventing the alteration caused by oxidative stress [31]because they scavenge and suppress the formation of free rad-icals Thus the use of preparations with antioxidant activity iscritical for cells that are in oxidative stress conditions

34 The Antioxidant Activity of Rosmarinic Acid in NormalConditions and under Oxidative Stress In order to evaluatethe antioxidant activity of different concentrations of RAin normal conditions and under oxidative stress in thenext series of experiments we measured the quantity ofthe intracellular ROS in cells using the DCFH-DA dye(Figures 3(a) and 3(b)) For these experiments we choseH2O2concentration of 100120583M because this is the lowest

concentration which after 24 hours statically significantlyreduces cell viability It is known that H

2O2should relatively

easily diffuse intoout of the cell through lipid bilayer oraquaporins In our experiments (Figure 3(b)) this was clearlyvisible since the amount of intracellular ROS in cells thatwere affected by 100 120583M of H

2O2during the original period

(05 h) increased by 30 whereas in control cells (withoutH2O2) the amount of ROS increased by an average of 7ndash

10 The lowest amount of RA (005mgmL) that affectedkeratinocyte cells only slightly reduced the amount of ROS(by 43) but all the higher amounts of RA that were used in

Evidence-Based Complementary and Alternative Medicine 5

Cel

l via

bilit

y (

)120

100

80

60

40

20

0Control 50 100 150 200 300 400 500

lowast

lowast

lowast

lowast

lowast

lowast

(120583M H2O2)

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0

Control

100 150 200

lowast

lowast

lowast

lowast

lowast

lowast

H2O2RA 005mg and H2O2

RA 025mg and H2O2RA 05mg and H2O2

(120583M H2O2)

(b)

Figure 2 Effect of different concentrations ofH2O2andRAonHaCaT cell viability HaCaT cells were treatedwith (a) different concentrations

(50ndash500 120583M) of H2O2for 24 hours and (b) different concentrations (005ndash05mgmL) of RA and H

2O2(100ndash200 120583M) for 24 hours Cell

viability was assessed using the MTT method Data are presented as means of the percentage of the untreated control cells plusmn SE (119899 = 4)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

4

2

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

(a)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

Control + 100120583M H2O2

(b)

Figure 3 Effects of different concentrations of rosmarinic acid on intracellular ROS generation in HaCaT cells Cells were preincubated withDCFH-DA (10 120583M) for 30min and then washed twice and (a) treated with different concentrations of RA and (b) treated with 100120583MH

2O2

and different concentrations of RA Control cells were treated with the same amounts of the solvent The number of experiments is 3

the study in all the studied time periods significantly reducedintracellular ROS quantity After 3 hours the amount of ROSwas below 14ndash28 at 01ndash05mgmL of RA In cells affected by05mg of RA the amount of ROS was close to that observedin the control cells

The skin is the interface between the body and itsenvironment and acts as a barrier protecting from adverseexternal factors Keratinocytes are the principal cell typecomprising the epidermis and constituting 90 of the total

amount of epidermal cells [32] For this reason the humankeratinocyte cell line HaCaT that was used in this researchwas an excellent model to evaluate the biological effect ofthe active substance of themodeled preparations Chemicallyrosmarinic acid is a derivative of two connected phenolicacids (coffee and 34-dihydroxyphenyl lactic acid) and has4 OH groups There are studies showing that it is the mostpotent antioxidant among the hydroxycinnamic acids [33]Studies have shown that rosmarinic acid is an effective

6 Evidence-Based Complementary and Alternative Medicine

scavenger of the DPPH radical and it also scavenges thereactive nitrogen species peroxynitrite and various ROS[34] The results of experiments done with cell cultures showthat through its antioxidant activity RA could be able toattenuate H

2O2-induced cell injury [35] The results of our

experiments on skin cells also demonstrated that RA haddose-dependent antioxidant activity In normal conditions05mgmL of RA after 3 hours reduced the amount ofintracellular ROS to 23 (Figure 3(a)) Therefore in normalconditions the examined preparations can provide protectiveand antiaging effects Scientific data confirmed that manyexternal factors (such as UV light traumas ultrasoundinfections air pollutants and cigarette smoke) and internalfactors (drugs contaminated food and some diseases suchas atopic dermatitis) cause increased ROS levels in skin cellsMost of the ROS have a short life span and are immobile (eghydroxyl or superoxide radicals) and they immediately (atthe source) react with the surrounding biomolecules H

2O2

is stable and easily diffusible in an aqueous environment Iteasily passes through biological membranes and therefore issuitable to create oxidative stress in experimental conditionsCell viability-decreasing H

2O2concentrations determined in

our experiments with HaCaT cells (EC501837 plusmn 204) were

similar to those found by Liu et al [22] Investigations of otherauthors demonstrate that the first signs of cell impairmentare noted at 50 120583M H

2O2 On the other hand 150 120583M of

H2O2and 300 120583MofH

2O2are relatively high doses to induce

apoptosis in Jurkat T cells and in HaCaT cells respectively[32 36] It was shown that very severe oxidative stressactivates a large number of signaling pathways in cells andcauses cell death via either apoptotic or necrotic mechanisms[37] The RA concentration of 005mgmL slightly reducedthe amount of intracellular ROS and had only limited effecton the oxidative state of cells Higher amounts of RA (01and 025mgmL) decreased the level of ROS by 14 and 22respectively However the amount of radicals in cells wasstill higher than that in the controls In normal conditionsskin cells do not sustain that much damage from harmfulenvironmental factors to initiate the production of such largeamounts of ROS that they would cause cell death Only with05mgmL of RA a decrease in the amount of ROS by 28was achieved and the remaining amount of ROS was similarto that in the control cells Based on these findings it is clearthat preparations with lemon balm extract can reduce theamount of ROS increased because of unfavorable externalconditions the effect of internal factors on the body or apathologic skin process in intracellular keratinocytes

4 Conclusions

Lemon balm hydrogels were modeled using carbomermethylcellulose and mixtures of them as gelling materialsThe study showed that the used gelling material affected thephysicochemical properties of semisolid preparations Theresults of biopharmaceutical tests in vitro showed that eventhough the highest amount of released RA was found in themethylcellulose-based gels carbomer (up to 10) due to itssemisolid consistency was a more appropriate base for theinsertion of lemon balm extracts The results of biological

investigations showed that rosmarinic acid depending on theconcentration reduced the amount of intracellular ROS inhuman keratinocyte cells and enhanced cell viability underoxidative stress

Conflict of Interests

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

References

[1] R Vejdani H R M Shalmani M Mir-Fattahi et al ldquoTheefficacy of an herbal medicine Carmint on the relief ofabdominal pain and bloating in patients with irritable bowelsyndrome a pilot studyrdquoDigestive Diseases and Sciences vol 51no 8 pp 1501ndash1507 2006

[2] J Lee E Jung J Koh Y S Kim and D Park ldquoEffect ofrosmarinic acid on atopic dermatitisrdquo Journal of Dermatologyvol 35 no 12 pp 768ndash771 2008

[3] E N Martins N T C Pessano L Leal et al ldquoProtectiveeffect of Melissa officinalis aqueous extract against Mn-inducedoxidative stress in chronically exposed micerdquo Brain ResearchBulletin vol 87 no 1 pp 74ndash79 2012

[4] H Moradkhani E Sargsyan H Bibak et al ldquoMelissa officinalisL a valuable medicine plant a reviewrdquo Journal of MedicinalPlants Research vol 4 no 25 pp 2753ndash2759 2010

[5] A Zarei S Changizi-Ashtiyani S Taheri and N Hosseini ldquoAbrief overviewof the effects ofMelissa officinalisL extract on thefunction of various body organsrdquo Zahedan Journal of Researchin Medical Sciences vol 15 pp 29ndash34 2015

[6] A Arceusz andMWesolowski ldquoQuality consistency evaluationof Melissa officinalis L commercial herbs by HPLC fingerprintand quantitation of selected phenolic acidsrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 83 pp 215ndash220 2013

[7] A Astani J Reichling and P Schnitzler ldquoMelissa officinalisextract inhibits attachment of herpes simplex virus in vitrordquoChemotherapy vol 58 no 1 pp 70ndash77 2012

[8] R Awad A Muhammad T Durst V L Trudeau and JT Arnason ldquoBioassay-guided fractionation of lemon balm(Melissa officinalis L) using an in vitro measure of GABAtransaminase activityrdquo Phytotherapy Research vol 23 no 8 pp1075ndash1081 2009

[9] A E Ince S Sahin and S G Sumnu ldquoExtraction of phenoliccompounds from melissa using microwave and ultrasoundrdquoTurkish Journal of Agriculture and Forestry vol 37 no 1 pp 69ndash75 2013

[10] G Mazzanti L Battinelli C Pompeo et al ldquoInhibitory activityof Melissa officinalis L extract on Herpes simplex virus type 2replicationrdquo Natural Product Research vol 22 no 16 pp 1433ndash1440 2008

[11] MMorrison LemonBalm httpwwwherballegacycomMor-rison Lemon Balmhtml

[12] A Bounihi GHajjaj R Alnamer Y Cherrah andA Zellou ldquoInvivo potential anti-inflammatory activity of Melissa officinalisL essential oilrdquoAdvances in Pharmacological Sciences vol 2013Article ID 101759 7 pages 2013

[13] M A Ribeiro M G Bernardo-Gil and M M EsquıvelldquoMelissa officinalis L study of antioxidant activity in supercrit-ical residuesrdquo Journal of Supercritical Fluids vol 21 no 1 pp51ndash60 2001

Evidence-Based Complementary and Alternative Medicine 7

[14] M Foldvari ldquoNon-invasive administration of drugs throughthe skin challenges in delivery system designrdquo PharmaceuticalScience amp Technology Today vol 3 no 12 pp 417ndash425 2000

[15] AVerma S Singh R Kaur A Kumar andUK Jain ldquoFormula-tion optimization and evaluation of clobetasol propionate gelrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 666ndash674 2013

[16] M G Dodov K Goracinova M Simonoska S Trajkovic-Jolevska J T Ribarska and M D Mitevska ldquoFormulation andevaluation of diazepamhydrogel for rectal administrationrdquoActaPharmaceutica vol 55 no 3 pp 251ndash261 2005

[17] FDAGuidance for Industry Nonsterile Semisolid Dosage FormsScale-Up and Postapproval Changes Chemistry Manufacturingand Controls In Vitro Release Testing and In Vivo BioequivalenceDocumentation FDA 1997

[18] G L Flynn V P Shah S N Tenjarla et al ldquoAssessment of valueand applications of in vitro testing of topical dermatologicaldrug productsrdquoPharmaceutical Research vol 16 no 9 pp 1325ndash1330 1999

[19] A Stelmakiene K Ramanauskiene and V Briedis ldquoRelease ofrosmarinic acid from semisolid formulations and its penetra-tion through human skin ex vivordquo Acta Pharmaceutica vol 65no 2 pp 199ndash205 2015

[20] M Zilius K Ramanauskiene and V Briedis ldquoRelease ofpropolis phenolic acids from semisolid formulations and theirpenetration into the human skin in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID958717 7 pages 2013

[21] F J Ahmad M A Alam Z I Khan R K Khar and MAli ldquoDevelopment and in vitro evaluation of an acid bufferingbioadhesive vaginal gel for mixed vaginal infectionsrdquo ActaPharmaceutica vol 58 no 4 pp 407ndash419 2008

[22] L Liu H Xie X Chen et al ldquoDifferential response of normalhuman epidermal keratinocytes and HaCaT cells to hydrogenperoxide-induced oxidative stressrdquo Clinical amp ExperimentalDermatology vol 37 no 7 pp 772ndash780 2012

[23] V Armoskaite K Ramanauskiene and V Briedis ldquoEvaluationof base for optimal drug delivery for iontophoretic therapyinvestigation of quality and stabilityrdquo African Journal of Phar-macy and Pharmacology vol 6 pp 1685ndash1695 2012

[24] M-H Schmid-Wendtner and H C Korting ldquoThe pH of theskin surface and its impact on the barrier functionrdquo SkinPharmacology and Physiology vol 19 no 6 pp 296ndash302 2006

[25] K Rizi R J Green M X Donaldson and A C WilliamsldquoUsing pH abnormalities in diseased skin to trigger and targettopical therapyrdquo Pharmaceutical Research vol 28 no 10 pp2589ndash2598 2011

[26] K Cal ldquoAcross skin barrier known methods new perfor-mancesrdquo Frontiers in Drug Design amp Discovery vol 4 no 1 pp162ndash188 2009

[27] D R Drake K A Brogden D V Dawson and P W WertzldquoThematic review series skin lipids Antimicrobial lipids at theskin surfacerdquo The Journal of Lipid Research vol 49 no 1 pp4ndash11 2008

[28] G P Moss J C Dearden H Patel and M T D CroninldquoQuantitative structure-permeability relationships (QSPRs) forpercutaneous absorptionrdquo Toxicology in Vitro vol 16 no 3 pp299ndash317 2002

[29] D Touboul F Halgand A Brunelle et al ldquoTissue molecularion imaging by gold cluster ion bombardmentrdquo AnalyticalChemistry vol 76 no 6 pp 1550ndash1559 2004

[30] N Sivaranjani S V Rao andG Rajeev ldquoRole of reactive oxygenspecies and antioxidants in atopic dermatitisrdquo Journal of Clinicaland Diagnostic Research vol 7 no 12 pp 2683ndash2685 2013

[31] O Fadel K El Kirat and S Morandat ldquoThe natural antioxidantrosmarinic acid spontaneously penetratesmembranes to inhibitlipid peroxidation in siturdquo Biochimica et Biophysica ActamdashBiomembranes vol 1808 no 12 pp 2973ndash2980 2011

[32] E-R Lee Y-J Kang J-H KimH T Lee and S-G Cho ldquoMod-ulation of apoptosis in HaCaT keratinocytes via differentialregulation of ERK signaling pathway by flavonoidsrdquoThe Journalof Biological Chemistry vol 280 no 36 pp 31498ndash31507 2005

[33] M A Soobrattee V S Neergheen A Luximon-Ramma OI Aruoma and T Bahorun ldquoPhenolics as potential antiox-idant therapeutic agents Mechanism and actionsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

[34] NAKelseyHMWilkins andDA Linseman ldquoNutraceuticalantioxidants as novel neuroprotective agentsrdquoMolecules vol 15no 11 pp 7792ndash7814 2010

[35] L P Gao H L Wei H S Zhao S Y Xiao and R-L ZhengldquoAntiapoptotic and antioxidant effects of rosmarinic acid inastrocytesrdquo Pharmazie vol 60 no 1 pp 62ndash65 2005

[36] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[37] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

Submit your manuscripts athttpwwwhindawicom

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Composition of experimental lemon balm hydrogels

Composition () GelsN1 N2 N3 N4 N5 N6 N7 N8 N9

Carbomer 980 05 10 15 05 05 05 mdash mdash mdashMethylcellulose 15cP mdash mdash mdash 10 20 40 10 20 40Propylene glycol 200 200 200 200 200 200 200 200 20010 NaOH qs ad pH 7 qs ad pH 7 qs ad pH 7 qs ad pH 7 qs ad pH 7 qs ad pH 7 mdash mdash mdashMelissa officinalis (L)dry extract 40 40 40 40 40 40 40 40 40

Purified qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000 qs ad 1000

therapeutic effects of the final product [14] A suitablebase ensures the stability of semisolid preparations duringstorage good distribution on the skin and an efficient releaseof a drug substance from the preparation [14] Creams andhydrophilic gels are the most common topical preparationsapplied on the skin due to their positive sensory propertiesThe advantage that gel has over ointments or cream is that itis a more stable nonslimy and semisolid form of medication[15] Due to its characteristic sensory properties (easilyapplicable on the skin easily cleaned with water and leavingno greasy membrane on skin unlike other ointments) gelis suitable for the modeling of natural preparations thatare usually applied several times a day for longer periodsof time Gel ensures easy and safe administration of suchpreparations at home by nonmedical persons [16] Becauseof the aforementioned properties it is important to producegel containing lemon balm extract and to assess its qualityusing biopharmaceutical tests Quality control testing ofsuch topical semisolid dosage forms includes the identityquantitative analysis homogeneity rheological propertiesparticle size determination consistency and medicinalsubstance release tests (Eur Ph 60 0120080132) Invitro release testing identifies the influence of a number ofphysicochemical parameters such as solubility of the drugsubstance in the carrier or the effect of particle size andviscosity of the dosage form on the release of the medicinalsubstance from the dosage form through a syntheticmembrane into the acceptor medium [17 18] The resultsof scientific research revealed that rosmarinic acid is moreeasily released from emulsion bases than from ointments[19] Because of the lack of data of biopharmaceuticalscientific tests it is important to assess the rate of RA releasefrom modulated hydrophilic gels

The aim of the study was to design pharmaceuticalhydrophilic semisolid dosage forms with lemon balm extractassess their quality and investigate the protective effect ofthe main active substance (rosmarinic acid) on skin cells innormal conditions and under oxidative stress

2 Materials and Methods

Experimental lemon balm hydrogel formulations were pre-pared according to the general technological principles ofsemisolid preparations [20] The compositions of the formu-lated gels are presented in Table 1 These gels were developed

to study the effect of individual gelling polymers on drugrelease behavior Dry lemon balm extract was dissolved inwater and dispersed in swelled gels under stirring Carbomergels (N1ndashN3) were prepared by neutralization with sodiumhydroxide Gels of methylcellulose (N7ndashN9) were preparedby overnight soaking of the polymer for complete hydrationCombination gels (N4ndashN6) were prepared by mixing poly-mer gels together [21]

21 Physicochemical and Biopharmaceutical Properties of GelsThe dynamic viscosity of gels was determined at room tem-perature using a Vibro Viscometer SV-10 (A amp D CompanyLtd Japan) The studied substance was placed into a specialcontainer for measurement Subsequently the container wasfixed on the working surface of the device and sensors weresubmerged into the studied substance The rotation speed ofa cylindrical spindle was 100 revmin The length of everymeasurement was 10 sec (119899 = 3)

The pH of semisolid preparations was analyzed using apH meter HD 21051 (Delta OHM Italy) A 5 solution wasprepared to determine pH levels The appropriate amount ofthe semisolid formula was topped with purified water andstirred for 30minutes on an IKAMAGC-MAGHS7magneticstirrer (IKA-Werke GmbH amp Co KG Staufen Germany) ata temperature of 50∘C Then the solution was cooled andfiltered through a paper filter

In vitro release experiments were performed using mod-ified Franz-type diffusion cells The semisolid sample (100 plusmn002 g) was placed into the cell with a dialysis membraneThe dialysis membrane Cuprophan (Medicell InternationalLtd UK) was made of natural cellulose The area of thediffusion was 177 cm2 Purified water acted as the acceptormedium The temperature of the acceptor medium was keptat 37plusmn02∘CThemediumwas stirred using amagnetic stirrerSamples from the acceptor solution were taken at 1 2 4 and6 h and were immediately replaced with the same volume offresh acceptor solution

Lemon balm extracts were analyzed by applying high-performance liquid chromatography The capillary HPLCmethod was developed and validated The amount of theRA was evaluated by applying the validated HPLC methodusing the Agilent 1260 Infinity Capillary LC System (AgilentTechnologies USA) with an Agilent Diode Array Detec-tor The separation was performed in a C18 ACE (5120583m)150 times 05 id column The mobile phase consisted of solvents

Evidence-Based Complementary and Alternative Medicine 3

A (05 aqueous solution of acetic acid 119881119881) and B (ace-tonitrile) using the following gradient elution 23 of Bat 0min 40 of B at 10min and 70 of B at 11ndash15minit subsequently returned to the initial conditions with 10-minute reequilibration with the total run time of 25 minutesThe analysis was carried out at a flow rate of 10 120583Lminminus1 withthe detection wavelength set at 330 nm

22 Cell Lines and Cell Culture Human keratinocyte cell lineHaCaT was purchased from the Cell Lines Service GmbH(Germany) Cells of convenient concentration were seededin culture flasks containing DMEM with 10 of fetal bovineserum 100UmL penicillin and 100 120583gmL streptomycinCultures were then incubated at 37∘C with 5 CO

2and

saturated humidity culture transfer was performed once aweek and the medium was renewed twice a week

23 Measurement of Cell Viability Twenty-four hours priorto testing cells with the investigated preparations were trans-ferred to 96-well plates at concentrations of 30000 cellswellThe cells were incubated with (A) different concentrationsof H2O2and (B) different concentrations of RA and H

2O2

After 24-hour treatment with the preparations the DMEMmedium was removed and the cells were washed twice with100mLwell warm Phosphate Buffered Saline (PBS) Afterwashing 180 120583Lwell PBS was added along with 20120583Lwellof 5mgmLMTT dye dissolved in PBS to each well The cellswere incubatedwithMTT at 37∘C for 2 hours Afterwards thedyewas removed the intracellularly formed crystals were dis-solved in DMSO (100 120583Lwell) and the plate was kept in thedark for 15 minutes The absorption was measured at 570 nmand 620 nm as reference with a microplate spectrophotome-ter (Sunrise Tecan Group Ltd Switzerland)The results wereexpressed as a fluorescence percentage in control cells

24 Measurement of Intracellular ROS Generation The pro-duction of ROS was assessed using the 2101584071015840-dichlorofluores-cein diacetate (DCFH-DA) as described in [22] Twenty-fourhours prior to treating cells with RA they were transferredto 96-well plates at concentrations of 50000 cellswell After24 hours the medium was removed and cells were washedwith PBS After washing 200120583Lwell HBSS medium sup-plemented with DCFH-DA (10 120583M) was added The cellswere incubated at 37∘C for 30min After the incubation thecells were washed twice with PBS and were subsequentlytreated with (A) different concentrations (005ndash05mg) ofRA or (B) 100120583M of H

2O2and different concentrations of

RA The fluorescence of DCF was detected by a fluorometerat excitation and emission of 488 and 525 nm wavelengthsrespectively Data are presented as means of fluorescenceintensity plusmn SE

25 Statistical Analysis of the Results All tests were donein triplicate Mean values and standard deviations of theresults were calculated by using Microsoft Office Excel 2010and SigmaPlot version 120 (Systat Software Inc) softwareThe significance of differences in test results was assessed byusing Studentrsquos 119905-test The differences were considered to bestatistically significant when 119901 lt 005

3 Results and Discussion

31 Evaluation of Physicochemical Properties ofHydrogels Forsemisolid preparations that could be applied to the treat-ment of atopic dermatitis natural medicinal plant materialdry lemon balm extract containing 80mgg of rosmarinicacid was selected as the active substance Gels producedwith lemon balm extract were homogeneous and yellowishto brownish in color According to the results (Table 2)gels that were produced with carbomer and the mixtureof carbomer and methylcellulose [N1ndashN6] had higher pHvalues Meanwhile gels produced with the gelling materialof methylcellulose [N7ndashN9] had weakly acidic pH the valuesranged from 584 to 632 It was observed that when theconcentration of methylcellulose in these gels increased thepH value of the gels was also increasing and they became lessacidic

The results in Table 2 show that the selected gellingmaterial affected the viscosity of the formulated gels Gels[N1ndashN6] in which carbomer was used as the main gellingmaterial had a higher viscosity whereas methylcellulose-based gels [N7ndashN9] had the lowest viscosity The lattersemisolid systems were characterized by a liquid consistencyThis shows that carbomer is a stronger gelling materialcompared to methylcellulose The results confirmed the datapublished in scientific literature indicating that the concen-tration of gelling material has an influence on the viscosityof semisolid preparations that is increasing carbomer andmethylcellulose concentrations in semisolid preparationsincreases their dynamic viscosity [15 23] Gels [N1ndashN3] inwhich carbomer was used as the main gelling material hadthe highest viscosity whereas the lowest dynamic viscositywas found in methylcellulose-based gels [N7ndashN9] Based onscientific literature the pH value of dermatological prepara-tions is an important quality indicator In order to reach themaximum effect of these preparations their pH should beclose to that of the skin (54 to 59) [24] In patients withatopic dermatitis the pH of the skin may increase (60 to65) [25] The results of our study showed that the producedsemisolid preparations are suitable for use on the skin theyare homogeneous and have suitable pH values and acceptableorganoleptic properties

32 Release of Rosmarinic Acid from Hydrogels The resultsshowed that the selected gelling material affected the releaseof RA content from semisolid systems (Figure 1) Lower andslower RA content release was more observed in carbomerand carbomer-methylcellulose-based combination gels thanin the methylcellulose gels alone In the evaluation of gelscontaining carbomer [N1ndashN3] as the main gelling materialthe highest RA content was released after 2 hours afterwhich time a slowdown of the release was observed SlowerRA content release kinetics was observed in carbomer-methylcellulose-based gels [N4ndashN6]The highest RA contentwas released after 4 hours and then the release sloweddown The highest RA content was released from gels inwhich methylcellulose was used as the gelling material Dataof the statistical analysis showed a statistically significantdifference (119901 lt 005) between RA quantities released from

4 Evidence-Based Complementary and Alternative Medicine

Table 2 Results of quality control testing in gels

Quality parameter GelsN1 N2 N3 N4 N5 N6 N7 N8 N9

pH value 745 plusmn 022 762 plusmn 031 775 plusmn 018 735 plusmn 021 720 plusmn 019 708 plusmn 013 584 plusmn 015 614 plusmn 027 632 plusmn 030Dynamic viscosity Pa s 245 plusmn 012 351 plusmn 008 386 plusmn 010 264 plusmn 014 290 plusmn 008 298 plusmn 003 001 plusmn 001 007 plusmn 001 061 plusmn 008

02468

101214161820

N1 N2 N3 N4 N5 N6 N7 N8 N9Samples

Rosm

arin

ic ac

id co

nten

t (

)

1h2h

4h6h

Figure 1 The kinetics of rosmarinic acid release from gels

methylcellulose-based hydrogels Even though the highestreleased RA content was found inmethylcellulose-based gelscarbomer due to its semisolid consistency is a more appro-priate base for the insertion of liquid lemon balm extractsMeanwhile methylcellulose can be used for the productionof gels in order to achieve a prolonged action of themedicinalpreparation Biopharmaceutical research proved that whenthe concentration of gelling materials in semisolid dosageforms is increasing the released RA content is decreasingIn vitro study of released RA is an informative tool for theassessment of the suitability of the base as a carrier for theinsertion of liquid lemon balm extract Scientific studieshave shown that RA was accumulating in the epidermis andthe hydrophilic dermis acted as a barrier preventing deeperpenetration and entry of RA into systemic blood flow [19]Substance penetration into or through the skin occurs in acoherent sequence when dissolved molecules released fromthe dosage form reach the surface of the stratumcorneumandpenetrate through it [26] Materials can penetrate throughthe skin by intracellular extracellular and additional ldquoshuntrdquo(through hair follicles and gland ducts) routes [27 28]Substance penetration using the intracellular polar routeoccurswhenmolecules diffuse through the cytoplasmof deadkeratinocytes and the surrounding lipid matrix [29] For thisreason the human keratinocyte cell line HaCaT was chosenfor further studies of the biological effects of the releasedRA With these studies we aimed at determining whetherthe RA released from the gel has protective effect in normalconditions and under oxidative stress

33 Influence of H2O2 and Rosmarinic Acid on HaCaTCell Viability during H2O2 Exposure In order to assess theeffect of different concentrations of H

2O2on HaCaT cell

viability the MTT assay was carried out The results of theseexperiments are presented in Figure 2(a) EC

50was estimated

to be 1837 plusmn 204 120583MH2O2

For further experiments we chose RA concentrations(005ndash05mgmL) according to the results of RA release fromthe gel received at various time periods and incubated thecells for 24 hours The results of our experiments revealed(Figure 2(b)) that higher concentrations of RA (025ndash05mgmL) protected the cells from the damage caused bydifferent concentrations of H

2O2and enhanced cell viability

by 10ndash24 Moreover 05mgmL of RA restored HaCaT cellviability until the control level in the presence of 100120583M ofH2O2

It is possible that the protective effect of RA is relatedto its antioxidant activity Oxidative stress conditions in cellsdevelop in the presence of an imbalance between ROS gen-eration and endogenous antioxidant defense mechanismsThis state of oxidative stress can affect all important cellularcomponents like proteins DNA andmembrane lipids whichis considered to be the main mechanism of cell damage[30] There is research confirming that exogenous moleculesfrom dietary sources such as polyphenols are very efficientin preventing the alteration caused by oxidative stress [31]because they scavenge and suppress the formation of free rad-icals Thus the use of preparations with antioxidant activity iscritical for cells that are in oxidative stress conditions

34 The Antioxidant Activity of Rosmarinic Acid in NormalConditions and under Oxidative Stress In order to evaluatethe antioxidant activity of different concentrations of RAin normal conditions and under oxidative stress in thenext series of experiments we measured the quantity ofthe intracellular ROS in cells using the DCFH-DA dye(Figures 3(a) and 3(b)) For these experiments we choseH2O2concentration of 100120583M because this is the lowest

concentration which after 24 hours statically significantlyreduces cell viability It is known that H

2O2should relatively

easily diffuse intoout of the cell through lipid bilayer oraquaporins In our experiments (Figure 3(b)) this was clearlyvisible since the amount of intracellular ROS in cells thatwere affected by 100 120583M of H

2O2during the original period

(05 h) increased by 30 whereas in control cells (withoutH2O2) the amount of ROS increased by an average of 7ndash

10 The lowest amount of RA (005mgmL) that affectedkeratinocyte cells only slightly reduced the amount of ROS(by 43) but all the higher amounts of RA that were used in

Evidence-Based Complementary and Alternative Medicine 5

Cel

l via

bilit

y (

)120

100

80

60

40

20

0Control 50 100 150 200 300 400 500

lowast

lowast

lowast

lowast

lowast

lowast

(120583M H2O2)

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0

Control

100 150 200

lowast

lowast

lowast

lowast

lowast

lowast

H2O2RA 005mg and H2O2

RA 025mg and H2O2RA 05mg and H2O2

(120583M H2O2)

(b)

Figure 2 Effect of different concentrations ofH2O2andRAonHaCaT cell viability HaCaT cells were treatedwith (a) different concentrations

(50ndash500 120583M) of H2O2for 24 hours and (b) different concentrations (005ndash05mgmL) of RA and H

2O2(100ndash200 120583M) for 24 hours Cell

viability was assessed using the MTT method Data are presented as means of the percentage of the untreated control cells plusmn SE (119899 = 4)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

4

2

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

(a)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

Control + 100120583M H2O2

(b)

Figure 3 Effects of different concentrations of rosmarinic acid on intracellular ROS generation in HaCaT cells Cells were preincubated withDCFH-DA (10 120583M) for 30min and then washed twice and (a) treated with different concentrations of RA and (b) treated with 100120583MH

2O2

and different concentrations of RA Control cells were treated with the same amounts of the solvent The number of experiments is 3

the study in all the studied time periods significantly reducedintracellular ROS quantity After 3 hours the amount of ROSwas below 14ndash28 at 01ndash05mgmL of RA In cells affected by05mg of RA the amount of ROS was close to that observedin the control cells

The skin is the interface between the body and itsenvironment and acts as a barrier protecting from adverseexternal factors Keratinocytes are the principal cell typecomprising the epidermis and constituting 90 of the total

amount of epidermal cells [32] For this reason the humankeratinocyte cell line HaCaT that was used in this researchwas an excellent model to evaluate the biological effect ofthe active substance of themodeled preparations Chemicallyrosmarinic acid is a derivative of two connected phenolicacids (coffee and 34-dihydroxyphenyl lactic acid) and has4 OH groups There are studies showing that it is the mostpotent antioxidant among the hydroxycinnamic acids [33]Studies have shown that rosmarinic acid is an effective

6 Evidence-Based Complementary and Alternative Medicine

scavenger of the DPPH radical and it also scavenges thereactive nitrogen species peroxynitrite and various ROS[34] The results of experiments done with cell cultures showthat through its antioxidant activity RA could be able toattenuate H

2O2-induced cell injury [35] The results of our

experiments on skin cells also demonstrated that RA haddose-dependent antioxidant activity In normal conditions05mgmL of RA after 3 hours reduced the amount ofintracellular ROS to 23 (Figure 3(a)) Therefore in normalconditions the examined preparations can provide protectiveand antiaging effects Scientific data confirmed that manyexternal factors (such as UV light traumas ultrasoundinfections air pollutants and cigarette smoke) and internalfactors (drugs contaminated food and some diseases suchas atopic dermatitis) cause increased ROS levels in skin cellsMost of the ROS have a short life span and are immobile (eghydroxyl or superoxide radicals) and they immediately (atthe source) react with the surrounding biomolecules H

2O2

is stable and easily diffusible in an aqueous environment Iteasily passes through biological membranes and therefore issuitable to create oxidative stress in experimental conditionsCell viability-decreasing H

2O2concentrations determined in

our experiments with HaCaT cells (EC501837 plusmn 204) were

similar to those found by Liu et al [22] Investigations of otherauthors demonstrate that the first signs of cell impairmentare noted at 50 120583M H

2O2 On the other hand 150 120583M of

H2O2and 300 120583MofH

2O2are relatively high doses to induce

apoptosis in Jurkat T cells and in HaCaT cells respectively[32 36] It was shown that very severe oxidative stressactivates a large number of signaling pathways in cells andcauses cell death via either apoptotic or necrotic mechanisms[37] The RA concentration of 005mgmL slightly reducedthe amount of intracellular ROS and had only limited effecton the oxidative state of cells Higher amounts of RA (01and 025mgmL) decreased the level of ROS by 14 and 22respectively However the amount of radicals in cells wasstill higher than that in the controls In normal conditionsskin cells do not sustain that much damage from harmfulenvironmental factors to initiate the production of such largeamounts of ROS that they would cause cell death Only with05mgmL of RA a decrease in the amount of ROS by 28was achieved and the remaining amount of ROS was similarto that in the control cells Based on these findings it is clearthat preparations with lemon balm extract can reduce theamount of ROS increased because of unfavorable externalconditions the effect of internal factors on the body or apathologic skin process in intracellular keratinocytes

4 Conclusions

Lemon balm hydrogels were modeled using carbomermethylcellulose and mixtures of them as gelling materialsThe study showed that the used gelling material affected thephysicochemical properties of semisolid preparations Theresults of biopharmaceutical tests in vitro showed that eventhough the highest amount of released RA was found in themethylcellulose-based gels carbomer (up to 10) due to itssemisolid consistency was a more appropriate base for theinsertion of lemon balm extracts The results of biological

investigations showed that rosmarinic acid depending on theconcentration reduced the amount of intracellular ROS inhuman keratinocyte cells and enhanced cell viability underoxidative stress

Conflict of Interests

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

References

[1] R Vejdani H R M Shalmani M Mir-Fattahi et al ldquoTheefficacy of an herbal medicine Carmint on the relief ofabdominal pain and bloating in patients with irritable bowelsyndrome a pilot studyrdquoDigestive Diseases and Sciences vol 51no 8 pp 1501ndash1507 2006

[2] J Lee E Jung J Koh Y S Kim and D Park ldquoEffect ofrosmarinic acid on atopic dermatitisrdquo Journal of Dermatologyvol 35 no 12 pp 768ndash771 2008

[3] E N Martins N T C Pessano L Leal et al ldquoProtectiveeffect of Melissa officinalis aqueous extract against Mn-inducedoxidative stress in chronically exposed micerdquo Brain ResearchBulletin vol 87 no 1 pp 74ndash79 2012

[4] H Moradkhani E Sargsyan H Bibak et al ldquoMelissa officinalisL a valuable medicine plant a reviewrdquo Journal of MedicinalPlants Research vol 4 no 25 pp 2753ndash2759 2010

[5] A Zarei S Changizi-Ashtiyani S Taheri and N Hosseini ldquoAbrief overviewof the effects ofMelissa officinalisL extract on thefunction of various body organsrdquo Zahedan Journal of Researchin Medical Sciences vol 15 pp 29ndash34 2015

[6] A Arceusz andMWesolowski ldquoQuality consistency evaluationof Melissa officinalis L commercial herbs by HPLC fingerprintand quantitation of selected phenolic acidsrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 83 pp 215ndash220 2013

[7] A Astani J Reichling and P Schnitzler ldquoMelissa officinalisextract inhibits attachment of herpes simplex virus in vitrordquoChemotherapy vol 58 no 1 pp 70ndash77 2012

[8] R Awad A Muhammad T Durst V L Trudeau and JT Arnason ldquoBioassay-guided fractionation of lemon balm(Melissa officinalis L) using an in vitro measure of GABAtransaminase activityrdquo Phytotherapy Research vol 23 no 8 pp1075ndash1081 2009

[9] A E Ince S Sahin and S G Sumnu ldquoExtraction of phenoliccompounds from melissa using microwave and ultrasoundrdquoTurkish Journal of Agriculture and Forestry vol 37 no 1 pp 69ndash75 2013

[10] G Mazzanti L Battinelli C Pompeo et al ldquoInhibitory activityof Melissa officinalis L extract on Herpes simplex virus type 2replicationrdquo Natural Product Research vol 22 no 16 pp 1433ndash1440 2008

[11] MMorrison LemonBalm httpwwwherballegacycomMor-rison Lemon Balmhtml

[12] A Bounihi GHajjaj R Alnamer Y Cherrah andA Zellou ldquoInvivo potential anti-inflammatory activity of Melissa officinalisL essential oilrdquoAdvances in Pharmacological Sciences vol 2013Article ID 101759 7 pages 2013

[13] M A Ribeiro M G Bernardo-Gil and M M EsquıvelldquoMelissa officinalis L study of antioxidant activity in supercrit-ical residuesrdquo Journal of Supercritical Fluids vol 21 no 1 pp51ndash60 2001

Evidence-Based Complementary and Alternative Medicine 7

[14] M Foldvari ldquoNon-invasive administration of drugs throughthe skin challenges in delivery system designrdquo PharmaceuticalScience amp Technology Today vol 3 no 12 pp 417ndash425 2000

[15] AVerma S Singh R Kaur A Kumar andUK Jain ldquoFormula-tion optimization and evaluation of clobetasol propionate gelrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 666ndash674 2013

[16] M G Dodov K Goracinova M Simonoska S Trajkovic-Jolevska J T Ribarska and M D Mitevska ldquoFormulation andevaluation of diazepamhydrogel for rectal administrationrdquoActaPharmaceutica vol 55 no 3 pp 251ndash261 2005

[17] FDAGuidance for Industry Nonsterile Semisolid Dosage FormsScale-Up and Postapproval Changes Chemistry Manufacturingand Controls In Vitro Release Testing and In Vivo BioequivalenceDocumentation FDA 1997

[18] G L Flynn V P Shah S N Tenjarla et al ldquoAssessment of valueand applications of in vitro testing of topical dermatologicaldrug productsrdquoPharmaceutical Research vol 16 no 9 pp 1325ndash1330 1999

[19] A Stelmakiene K Ramanauskiene and V Briedis ldquoRelease ofrosmarinic acid from semisolid formulations and its penetra-tion through human skin ex vivordquo Acta Pharmaceutica vol 65no 2 pp 199ndash205 2015

[20] M Zilius K Ramanauskiene and V Briedis ldquoRelease ofpropolis phenolic acids from semisolid formulations and theirpenetration into the human skin in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID958717 7 pages 2013

[21] F J Ahmad M A Alam Z I Khan R K Khar and MAli ldquoDevelopment and in vitro evaluation of an acid bufferingbioadhesive vaginal gel for mixed vaginal infectionsrdquo ActaPharmaceutica vol 58 no 4 pp 407ndash419 2008

[22] L Liu H Xie X Chen et al ldquoDifferential response of normalhuman epidermal keratinocytes and HaCaT cells to hydrogenperoxide-induced oxidative stressrdquo Clinical amp ExperimentalDermatology vol 37 no 7 pp 772ndash780 2012

[23] V Armoskaite K Ramanauskiene and V Briedis ldquoEvaluationof base for optimal drug delivery for iontophoretic therapyinvestigation of quality and stabilityrdquo African Journal of Phar-macy and Pharmacology vol 6 pp 1685ndash1695 2012

[24] M-H Schmid-Wendtner and H C Korting ldquoThe pH of theskin surface and its impact on the barrier functionrdquo SkinPharmacology and Physiology vol 19 no 6 pp 296ndash302 2006

[25] K Rizi R J Green M X Donaldson and A C WilliamsldquoUsing pH abnormalities in diseased skin to trigger and targettopical therapyrdquo Pharmaceutical Research vol 28 no 10 pp2589ndash2598 2011

[26] K Cal ldquoAcross skin barrier known methods new perfor-mancesrdquo Frontiers in Drug Design amp Discovery vol 4 no 1 pp162ndash188 2009

[27] D R Drake K A Brogden D V Dawson and P W WertzldquoThematic review series skin lipids Antimicrobial lipids at theskin surfacerdquo The Journal of Lipid Research vol 49 no 1 pp4ndash11 2008

[28] G P Moss J C Dearden H Patel and M T D CroninldquoQuantitative structure-permeability relationships (QSPRs) forpercutaneous absorptionrdquo Toxicology in Vitro vol 16 no 3 pp299ndash317 2002

[29] D Touboul F Halgand A Brunelle et al ldquoTissue molecularion imaging by gold cluster ion bombardmentrdquo AnalyticalChemistry vol 76 no 6 pp 1550ndash1559 2004

[30] N Sivaranjani S V Rao andG Rajeev ldquoRole of reactive oxygenspecies and antioxidants in atopic dermatitisrdquo Journal of Clinicaland Diagnostic Research vol 7 no 12 pp 2683ndash2685 2013

[31] O Fadel K El Kirat and S Morandat ldquoThe natural antioxidantrosmarinic acid spontaneously penetratesmembranes to inhibitlipid peroxidation in siturdquo Biochimica et Biophysica ActamdashBiomembranes vol 1808 no 12 pp 2973ndash2980 2011

[32] E-R Lee Y-J Kang J-H KimH T Lee and S-G Cho ldquoMod-ulation of apoptosis in HaCaT keratinocytes via differentialregulation of ERK signaling pathway by flavonoidsrdquoThe Journalof Biological Chemistry vol 280 no 36 pp 31498ndash31507 2005

[33] M A Soobrattee V S Neergheen A Luximon-Ramma OI Aruoma and T Bahorun ldquoPhenolics as potential antiox-idant therapeutic agents Mechanism and actionsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

[34] NAKelseyHMWilkins andDA Linseman ldquoNutraceuticalantioxidants as novel neuroprotective agentsrdquoMolecules vol 15no 11 pp 7792ndash7814 2010

[35] L P Gao H L Wei H S Zhao S Y Xiao and R-L ZhengldquoAntiapoptotic and antioxidant effects of rosmarinic acid inastrocytesrdquo Pharmazie vol 60 no 1 pp 62ndash65 2005

[36] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[37] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

A (05 aqueous solution of acetic acid 119881119881) and B (ace-tonitrile) using the following gradient elution 23 of Bat 0min 40 of B at 10min and 70 of B at 11ndash15minit subsequently returned to the initial conditions with 10-minute reequilibration with the total run time of 25 minutesThe analysis was carried out at a flow rate of 10 120583Lminminus1 withthe detection wavelength set at 330 nm

22 Cell Lines and Cell Culture Human keratinocyte cell lineHaCaT was purchased from the Cell Lines Service GmbH(Germany) Cells of convenient concentration were seededin culture flasks containing DMEM with 10 of fetal bovineserum 100UmL penicillin and 100 120583gmL streptomycinCultures were then incubated at 37∘C with 5 CO

2and

saturated humidity culture transfer was performed once aweek and the medium was renewed twice a week

23 Measurement of Cell Viability Twenty-four hours priorto testing cells with the investigated preparations were trans-ferred to 96-well plates at concentrations of 30000 cellswellThe cells were incubated with (A) different concentrationsof H2O2and (B) different concentrations of RA and H

2O2

After 24-hour treatment with the preparations the DMEMmedium was removed and the cells were washed twice with100mLwell warm Phosphate Buffered Saline (PBS) Afterwashing 180 120583Lwell PBS was added along with 20120583Lwellof 5mgmLMTT dye dissolved in PBS to each well The cellswere incubatedwithMTT at 37∘C for 2 hours Afterwards thedyewas removed the intracellularly formed crystals were dis-solved in DMSO (100 120583Lwell) and the plate was kept in thedark for 15 minutes The absorption was measured at 570 nmand 620 nm as reference with a microplate spectrophotome-ter (Sunrise Tecan Group Ltd Switzerland)The results wereexpressed as a fluorescence percentage in control cells

24 Measurement of Intracellular ROS Generation The pro-duction of ROS was assessed using the 2101584071015840-dichlorofluores-cein diacetate (DCFH-DA) as described in [22] Twenty-fourhours prior to treating cells with RA they were transferredto 96-well plates at concentrations of 50000 cellswell After24 hours the medium was removed and cells were washedwith PBS After washing 200120583Lwell HBSS medium sup-plemented with DCFH-DA (10 120583M) was added The cellswere incubated at 37∘C for 30min After the incubation thecells were washed twice with PBS and were subsequentlytreated with (A) different concentrations (005ndash05mg) ofRA or (B) 100120583M of H

2O2and different concentrations of

RA The fluorescence of DCF was detected by a fluorometerat excitation and emission of 488 and 525 nm wavelengthsrespectively Data are presented as means of fluorescenceintensity plusmn SE

25 Statistical Analysis of the Results All tests were donein triplicate Mean values and standard deviations of theresults were calculated by using Microsoft Office Excel 2010and SigmaPlot version 120 (Systat Software Inc) softwareThe significance of differences in test results was assessed byusing Studentrsquos 119905-test The differences were considered to bestatistically significant when 119901 lt 005

3 Results and Discussion

31 Evaluation of Physicochemical Properties ofHydrogels Forsemisolid preparations that could be applied to the treat-ment of atopic dermatitis natural medicinal plant materialdry lemon balm extract containing 80mgg of rosmarinicacid was selected as the active substance Gels producedwith lemon balm extract were homogeneous and yellowishto brownish in color According to the results (Table 2)gels that were produced with carbomer and the mixtureof carbomer and methylcellulose [N1ndashN6] had higher pHvalues Meanwhile gels produced with the gelling materialof methylcellulose [N7ndashN9] had weakly acidic pH the valuesranged from 584 to 632 It was observed that when theconcentration of methylcellulose in these gels increased thepH value of the gels was also increasing and they became lessacidic

The results in Table 2 show that the selected gellingmaterial affected the viscosity of the formulated gels Gels[N1ndashN6] in which carbomer was used as the main gellingmaterial had a higher viscosity whereas methylcellulose-based gels [N7ndashN9] had the lowest viscosity The lattersemisolid systems were characterized by a liquid consistencyThis shows that carbomer is a stronger gelling materialcompared to methylcellulose The results confirmed the datapublished in scientific literature indicating that the concen-tration of gelling material has an influence on the viscosityof semisolid preparations that is increasing carbomer andmethylcellulose concentrations in semisolid preparationsincreases their dynamic viscosity [15 23] Gels [N1ndashN3] inwhich carbomer was used as the main gelling material hadthe highest viscosity whereas the lowest dynamic viscositywas found in methylcellulose-based gels [N7ndashN9] Based onscientific literature the pH value of dermatological prepara-tions is an important quality indicator In order to reach themaximum effect of these preparations their pH should beclose to that of the skin (54 to 59) [24] In patients withatopic dermatitis the pH of the skin may increase (60 to65) [25] The results of our study showed that the producedsemisolid preparations are suitable for use on the skin theyare homogeneous and have suitable pH values and acceptableorganoleptic properties

32 Release of Rosmarinic Acid from Hydrogels The resultsshowed that the selected gelling material affected the releaseof RA content from semisolid systems (Figure 1) Lower andslower RA content release was more observed in carbomerand carbomer-methylcellulose-based combination gels thanin the methylcellulose gels alone In the evaluation of gelscontaining carbomer [N1ndashN3] as the main gelling materialthe highest RA content was released after 2 hours afterwhich time a slowdown of the release was observed SlowerRA content release kinetics was observed in carbomer-methylcellulose-based gels [N4ndashN6]The highest RA contentwas released after 4 hours and then the release sloweddown The highest RA content was released from gels inwhich methylcellulose was used as the gelling material Dataof the statistical analysis showed a statistically significantdifference (119901 lt 005) between RA quantities released from

4 Evidence-Based Complementary and Alternative Medicine

Table 2 Results of quality control testing in gels

Quality parameter GelsN1 N2 N3 N4 N5 N6 N7 N8 N9

pH value 745 plusmn 022 762 plusmn 031 775 plusmn 018 735 plusmn 021 720 plusmn 019 708 plusmn 013 584 plusmn 015 614 plusmn 027 632 plusmn 030Dynamic viscosity Pa s 245 plusmn 012 351 plusmn 008 386 plusmn 010 264 plusmn 014 290 plusmn 008 298 plusmn 003 001 plusmn 001 007 plusmn 001 061 plusmn 008

02468

101214161820

N1 N2 N3 N4 N5 N6 N7 N8 N9Samples

Rosm

arin

ic ac

id co

nten

t (

)

1h2h

4h6h

Figure 1 The kinetics of rosmarinic acid release from gels

methylcellulose-based hydrogels Even though the highestreleased RA content was found inmethylcellulose-based gelscarbomer due to its semisolid consistency is a more appro-priate base for the insertion of liquid lemon balm extractsMeanwhile methylcellulose can be used for the productionof gels in order to achieve a prolonged action of themedicinalpreparation Biopharmaceutical research proved that whenthe concentration of gelling materials in semisolid dosageforms is increasing the released RA content is decreasingIn vitro study of released RA is an informative tool for theassessment of the suitability of the base as a carrier for theinsertion of liquid lemon balm extract Scientific studieshave shown that RA was accumulating in the epidermis andthe hydrophilic dermis acted as a barrier preventing deeperpenetration and entry of RA into systemic blood flow [19]Substance penetration into or through the skin occurs in acoherent sequence when dissolved molecules released fromthe dosage form reach the surface of the stratumcorneumandpenetrate through it [26] Materials can penetrate throughthe skin by intracellular extracellular and additional ldquoshuntrdquo(through hair follicles and gland ducts) routes [27 28]Substance penetration using the intracellular polar routeoccurswhenmolecules diffuse through the cytoplasmof deadkeratinocytes and the surrounding lipid matrix [29] For thisreason the human keratinocyte cell line HaCaT was chosenfor further studies of the biological effects of the releasedRA With these studies we aimed at determining whetherthe RA released from the gel has protective effect in normalconditions and under oxidative stress

33 Influence of H2O2 and Rosmarinic Acid on HaCaTCell Viability during H2O2 Exposure In order to assess theeffect of different concentrations of H

2O2on HaCaT cell

viability the MTT assay was carried out The results of theseexperiments are presented in Figure 2(a) EC

50was estimated

to be 1837 plusmn 204 120583MH2O2

For further experiments we chose RA concentrations(005ndash05mgmL) according to the results of RA release fromthe gel received at various time periods and incubated thecells for 24 hours The results of our experiments revealed(Figure 2(b)) that higher concentrations of RA (025ndash05mgmL) protected the cells from the damage caused bydifferent concentrations of H

2O2and enhanced cell viability

by 10ndash24 Moreover 05mgmL of RA restored HaCaT cellviability until the control level in the presence of 100120583M ofH2O2

It is possible that the protective effect of RA is relatedto its antioxidant activity Oxidative stress conditions in cellsdevelop in the presence of an imbalance between ROS gen-eration and endogenous antioxidant defense mechanismsThis state of oxidative stress can affect all important cellularcomponents like proteins DNA andmembrane lipids whichis considered to be the main mechanism of cell damage[30] There is research confirming that exogenous moleculesfrom dietary sources such as polyphenols are very efficientin preventing the alteration caused by oxidative stress [31]because they scavenge and suppress the formation of free rad-icals Thus the use of preparations with antioxidant activity iscritical for cells that are in oxidative stress conditions

34 The Antioxidant Activity of Rosmarinic Acid in NormalConditions and under Oxidative Stress In order to evaluatethe antioxidant activity of different concentrations of RAin normal conditions and under oxidative stress in thenext series of experiments we measured the quantity ofthe intracellular ROS in cells using the DCFH-DA dye(Figures 3(a) and 3(b)) For these experiments we choseH2O2concentration of 100120583M because this is the lowest

concentration which after 24 hours statically significantlyreduces cell viability It is known that H

2O2should relatively

easily diffuse intoout of the cell through lipid bilayer oraquaporins In our experiments (Figure 3(b)) this was clearlyvisible since the amount of intracellular ROS in cells thatwere affected by 100 120583M of H

2O2during the original period

(05 h) increased by 30 whereas in control cells (withoutH2O2) the amount of ROS increased by an average of 7ndash

10 The lowest amount of RA (005mgmL) that affectedkeratinocyte cells only slightly reduced the amount of ROS(by 43) but all the higher amounts of RA that were used in

Evidence-Based Complementary and Alternative Medicine 5

Cel

l via

bilit

y (

)120

100

80

60

40

20

0Control 50 100 150 200 300 400 500

lowast

lowast

lowast

lowast

lowast

lowast

(120583M H2O2)

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0

Control

100 150 200

lowast

lowast

lowast

lowast

lowast

lowast

H2O2RA 005mg and H2O2

RA 025mg and H2O2RA 05mg and H2O2

(120583M H2O2)

(b)

Figure 2 Effect of different concentrations ofH2O2andRAonHaCaT cell viability HaCaT cells were treatedwith (a) different concentrations

(50ndash500 120583M) of H2O2for 24 hours and (b) different concentrations (005ndash05mgmL) of RA and H

2O2(100ndash200 120583M) for 24 hours Cell

viability was assessed using the MTT method Data are presented as means of the percentage of the untreated control cells plusmn SE (119899 = 4)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

4

2

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

(a)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

Control + 100120583M H2O2

(b)

Figure 3 Effects of different concentrations of rosmarinic acid on intracellular ROS generation in HaCaT cells Cells were preincubated withDCFH-DA (10 120583M) for 30min and then washed twice and (a) treated with different concentrations of RA and (b) treated with 100120583MH

2O2

and different concentrations of RA Control cells were treated with the same amounts of the solvent The number of experiments is 3

the study in all the studied time periods significantly reducedintracellular ROS quantity After 3 hours the amount of ROSwas below 14ndash28 at 01ndash05mgmL of RA In cells affected by05mg of RA the amount of ROS was close to that observedin the control cells

The skin is the interface between the body and itsenvironment and acts as a barrier protecting from adverseexternal factors Keratinocytes are the principal cell typecomprising the epidermis and constituting 90 of the total

amount of epidermal cells [32] For this reason the humankeratinocyte cell line HaCaT that was used in this researchwas an excellent model to evaluate the biological effect ofthe active substance of themodeled preparations Chemicallyrosmarinic acid is a derivative of two connected phenolicacids (coffee and 34-dihydroxyphenyl lactic acid) and has4 OH groups There are studies showing that it is the mostpotent antioxidant among the hydroxycinnamic acids [33]Studies have shown that rosmarinic acid is an effective

6 Evidence-Based Complementary and Alternative Medicine

scavenger of the DPPH radical and it also scavenges thereactive nitrogen species peroxynitrite and various ROS[34] The results of experiments done with cell cultures showthat through its antioxidant activity RA could be able toattenuate H

2O2-induced cell injury [35] The results of our

experiments on skin cells also demonstrated that RA haddose-dependent antioxidant activity In normal conditions05mgmL of RA after 3 hours reduced the amount ofintracellular ROS to 23 (Figure 3(a)) Therefore in normalconditions the examined preparations can provide protectiveand antiaging effects Scientific data confirmed that manyexternal factors (such as UV light traumas ultrasoundinfections air pollutants and cigarette smoke) and internalfactors (drugs contaminated food and some diseases suchas atopic dermatitis) cause increased ROS levels in skin cellsMost of the ROS have a short life span and are immobile (eghydroxyl or superoxide radicals) and they immediately (atthe source) react with the surrounding biomolecules H

2O2

is stable and easily diffusible in an aqueous environment Iteasily passes through biological membranes and therefore issuitable to create oxidative stress in experimental conditionsCell viability-decreasing H

2O2concentrations determined in

our experiments with HaCaT cells (EC501837 plusmn 204) were

similar to those found by Liu et al [22] Investigations of otherauthors demonstrate that the first signs of cell impairmentare noted at 50 120583M H

2O2 On the other hand 150 120583M of

H2O2and 300 120583MofH

2O2are relatively high doses to induce

apoptosis in Jurkat T cells and in HaCaT cells respectively[32 36] It was shown that very severe oxidative stressactivates a large number of signaling pathways in cells andcauses cell death via either apoptotic or necrotic mechanisms[37] The RA concentration of 005mgmL slightly reducedthe amount of intracellular ROS and had only limited effecton the oxidative state of cells Higher amounts of RA (01and 025mgmL) decreased the level of ROS by 14 and 22respectively However the amount of radicals in cells wasstill higher than that in the controls In normal conditionsskin cells do not sustain that much damage from harmfulenvironmental factors to initiate the production of such largeamounts of ROS that they would cause cell death Only with05mgmL of RA a decrease in the amount of ROS by 28was achieved and the remaining amount of ROS was similarto that in the control cells Based on these findings it is clearthat preparations with lemon balm extract can reduce theamount of ROS increased because of unfavorable externalconditions the effect of internal factors on the body or apathologic skin process in intracellular keratinocytes

4 Conclusions

Lemon balm hydrogels were modeled using carbomermethylcellulose and mixtures of them as gelling materialsThe study showed that the used gelling material affected thephysicochemical properties of semisolid preparations Theresults of biopharmaceutical tests in vitro showed that eventhough the highest amount of released RA was found in themethylcellulose-based gels carbomer (up to 10) due to itssemisolid consistency was a more appropriate base for theinsertion of lemon balm extracts The results of biological

investigations showed that rosmarinic acid depending on theconcentration reduced the amount of intracellular ROS inhuman keratinocyte cells and enhanced cell viability underoxidative stress

Conflict of Interests

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

References

[1] R Vejdani H R M Shalmani M Mir-Fattahi et al ldquoTheefficacy of an herbal medicine Carmint on the relief ofabdominal pain and bloating in patients with irritable bowelsyndrome a pilot studyrdquoDigestive Diseases and Sciences vol 51no 8 pp 1501ndash1507 2006

[2] J Lee E Jung J Koh Y S Kim and D Park ldquoEffect ofrosmarinic acid on atopic dermatitisrdquo Journal of Dermatologyvol 35 no 12 pp 768ndash771 2008

[3] E N Martins N T C Pessano L Leal et al ldquoProtectiveeffect of Melissa officinalis aqueous extract against Mn-inducedoxidative stress in chronically exposed micerdquo Brain ResearchBulletin vol 87 no 1 pp 74ndash79 2012

[4] H Moradkhani E Sargsyan H Bibak et al ldquoMelissa officinalisL a valuable medicine plant a reviewrdquo Journal of MedicinalPlants Research vol 4 no 25 pp 2753ndash2759 2010

[5] A Zarei S Changizi-Ashtiyani S Taheri and N Hosseini ldquoAbrief overviewof the effects ofMelissa officinalisL extract on thefunction of various body organsrdquo Zahedan Journal of Researchin Medical Sciences vol 15 pp 29ndash34 2015

[6] A Arceusz andMWesolowski ldquoQuality consistency evaluationof Melissa officinalis L commercial herbs by HPLC fingerprintand quantitation of selected phenolic acidsrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 83 pp 215ndash220 2013

[7] A Astani J Reichling and P Schnitzler ldquoMelissa officinalisextract inhibits attachment of herpes simplex virus in vitrordquoChemotherapy vol 58 no 1 pp 70ndash77 2012

[8] R Awad A Muhammad T Durst V L Trudeau and JT Arnason ldquoBioassay-guided fractionation of lemon balm(Melissa officinalis L) using an in vitro measure of GABAtransaminase activityrdquo Phytotherapy Research vol 23 no 8 pp1075ndash1081 2009

[9] A E Ince S Sahin and S G Sumnu ldquoExtraction of phenoliccompounds from melissa using microwave and ultrasoundrdquoTurkish Journal of Agriculture and Forestry vol 37 no 1 pp 69ndash75 2013

[10] G Mazzanti L Battinelli C Pompeo et al ldquoInhibitory activityof Melissa officinalis L extract on Herpes simplex virus type 2replicationrdquo Natural Product Research vol 22 no 16 pp 1433ndash1440 2008

[11] MMorrison LemonBalm httpwwwherballegacycomMor-rison Lemon Balmhtml

[12] A Bounihi GHajjaj R Alnamer Y Cherrah andA Zellou ldquoInvivo potential anti-inflammatory activity of Melissa officinalisL essential oilrdquoAdvances in Pharmacological Sciences vol 2013Article ID 101759 7 pages 2013

[13] M A Ribeiro M G Bernardo-Gil and M M EsquıvelldquoMelissa officinalis L study of antioxidant activity in supercrit-ical residuesrdquo Journal of Supercritical Fluids vol 21 no 1 pp51ndash60 2001

Evidence-Based Complementary and Alternative Medicine 7

[14] M Foldvari ldquoNon-invasive administration of drugs throughthe skin challenges in delivery system designrdquo PharmaceuticalScience amp Technology Today vol 3 no 12 pp 417ndash425 2000

[15] AVerma S Singh R Kaur A Kumar andUK Jain ldquoFormula-tion optimization and evaluation of clobetasol propionate gelrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 666ndash674 2013

[16] M G Dodov K Goracinova M Simonoska S Trajkovic-Jolevska J T Ribarska and M D Mitevska ldquoFormulation andevaluation of diazepamhydrogel for rectal administrationrdquoActaPharmaceutica vol 55 no 3 pp 251ndash261 2005

[17] FDAGuidance for Industry Nonsterile Semisolid Dosage FormsScale-Up and Postapproval Changes Chemistry Manufacturingand Controls In Vitro Release Testing and In Vivo BioequivalenceDocumentation FDA 1997

[18] G L Flynn V P Shah S N Tenjarla et al ldquoAssessment of valueand applications of in vitro testing of topical dermatologicaldrug productsrdquoPharmaceutical Research vol 16 no 9 pp 1325ndash1330 1999

[19] A Stelmakiene K Ramanauskiene and V Briedis ldquoRelease ofrosmarinic acid from semisolid formulations and its penetra-tion through human skin ex vivordquo Acta Pharmaceutica vol 65no 2 pp 199ndash205 2015

[20] M Zilius K Ramanauskiene and V Briedis ldquoRelease ofpropolis phenolic acids from semisolid formulations and theirpenetration into the human skin in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID958717 7 pages 2013

[21] F J Ahmad M A Alam Z I Khan R K Khar and MAli ldquoDevelopment and in vitro evaluation of an acid bufferingbioadhesive vaginal gel for mixed vaginal infectionsrdquo ActaPharmaceutica vol 58 no 4 pp 407ndash419 2008

[22] L Liu H Xie X Chen et al ldquoDifferential response of normalhuman epidermal keratinocytes and HaCaT cells to hydrogenperoxide-induced oxidative stressrdquo Clinical amp ExperimentalDermatology vol 37 no 7 pp 772ndash780 2012

[23] V Armoskaite K Ramanauskiene and V Briedis ldquoEvaluationof base for optimal drug delivery for iontophoretic therapyinvestigation of quality and stabilityrdquo African Journal of Phar-macy and Pharmacology vol 6 pp 1685ndash1695 2012

[24] M-H Schmid-Wendtner and H C Korting ldquoThe pH of theskin surface and its impact on the barrier functionrdquo SkinPharmacology and Physiology vol 19 no 6 pp 296ndash302 2006

[25] K Rizi R J Green M X Donaldson and A C WilliamsldquoUsing pH abnormalities in diseased skin to trigger and targettopical therapyrdquo Pharmaceutical Research vol 28 no 10 pp2589ndash2598 2011

[26] K Cal ldquoAcross skin barrier known methods new perfor-mancesrdquo Frontiers in Drug Design amp Discovery vol 4 no 1 pp162ndash188 2009

[27] D R Drake K A Brogden D V Dawson and P W WertzldquoThematic review series skin lipids Antimicrobial lipids at theskin surfacerdquo The Journal of Lipid Research vol 49 no 1 pp4ndash11 2008

[28] G P Moss J C Dearden H Patel and M T D CroninldquoQuantitative structure-permeability relationships (QSPRs) forpercutaneous absorptionrdquo Toxicology in Vitro vol 16 no 3 pp299ndash317 2002

[29] D Touboul F Halgand A Brunelle et al ldquoTissue molecularion imaging by gold cluster ion bombardmentrdquo AnalyticalChemistry vol 76 no 6 pp 1550ndash1559 2004

[30] N Sivaranjani S V Rao andG Rajeev ldquoRole of reactive oxygenspecies and antioxidants in atopic dermatitisrdquo Journal of Clinicaland Diagnostic Research vol 7 no 12 pp 2683ndash2685 2013

[31] O Fadel K El Kirat and S Morandat ldquoThe natural antioxidantrosmarinic acid spontaneously penetratesmembranes to inhibitlipid peroxidation in siturdquo Biochimica et Biophysica ActamdashBiomembranes vol 1808 no 12 pp 2973ndash2980 2011

[32] E-R Lee Y-J Kang J-H KimH T Lee and S-G Cho ldquoMod-ulation of apoptosis in HaCaT keratinocytes via differentialregulation of ERK signaling pathway by flavonoidsrdquoThe Journalof Biological Chemistry vol 280 no 36 pp 31498ndash31507 2005

[33] M A Soobrattee V S Neergheen A Luximon-Ramma OI Aruoma and T Bahorun ldquoPhenolics as potential antiox-idant therapeutic agents Mechanism and actionsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

[34] NAKelseyHMWilkins andDA Linseman ldquoNutraceuticalantioxidants as novel neuroprotective agentsrdquoMolecules vol 15no 11 pp 7792ndash7814 2010

[35] L P Gao H L Wei H S Zhao S Y Xiao and R-L ZhengldquoAntiapoptotic and antioxidant effects of rosmarinic acid inastrocytesrdquo Pharmazie vol 60 no 1 pp 62ndash65 2005

[36] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[37] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

Table 2 Results of quality control testing in gels

Quality parameter GelsN1 N2 N3 N4 N5 N6 N7 N8 N9

pH value 745 plusmn 022 762 plusmn 031 775 plusmn 018 735 plusmn 021 720 plusmn 019 708 plusmn 013 584 plusmn 015 614 plusmn 027 632 plusmn 030Dynamic viscosity Pa s 245 plusmn 012 351 plusmn 008 386 plusmn 010 264 plusmn 014 290 plusmn 008 298 plusmn 003 001 plusmn 001 007 plusmn 001 061 plusmn 008

02468

101214161820

N1 N2 N3 N4 N5 N6 N7 N8 N9Samples

Rosm

arin

ic ac

id co

nten

t (

)

1h2h

4h6h

Figure 1 The kinetics of rosmarinic acid release from gels

methylcellulose-based hydrogels Even though the highestreleased RA content was found inmethylcellulose-based gelscarbomer due to its semisolid consistency is a more appro-priate base for the insertion of liquid lemon balm extractsMeanwhile methylcellulose can be used for the productionof gels in order to achieve a prolonged action of themedicinalpreparation Biopharmaceutical research proved that whenthe concentration of gelling materials in semisolid dosageforms is increasing the released RA content is decreasingIn vitro study of released RA is an informative tool for theassessment of the suitability of the base as a carrier for theinsertion of liquid lemon balm extract Scientific studieshave shown that RA was accumulating in the epidermis andthe hydrophilic dermis acted as a barrier preventing deeperpenetration and entry of RA into systemic blood flow [19]Substance penetration into or through the skin occurs in acoherent sequence when dissolved molecules released fromthe dosage form reach the surface of the stratumcorneumandpenetrate through it [26] Materials can penetrate throughthe skin by intracellular extracellular and additional ldquoshuntrdquo(through hair follicles and gland ducts) routes [27 28]Substance penetration using the intracellular polar routeoccurswhenmolecules diffuse through the cytoplasmof deadkeratinocytes and the surrounding lipid matrix [29] For thisreason the human keratinocyte cell line HaCaT was chosenfor further studies of the biological effects of the releasedRA With these studies we aimed at determining whetherthe RA released from the gel has protective effect in normalconditions and under oxidative stress

33 Influence of H2O2 and Rosmarinic Acid on HaCaTCell Viability during H2O2 Exposure In order to assess theeffect of different concentrations of H

2O2on HaCaT cell

viability the MTT assay was carried out The results of theseexperiments are presented in Figure 2(a) EC

50was estimated

to be 1837 plusmn 204 120583MH2O2

For further experiments we chose RA concentrations(005ndash05mgmL) according to the results of RA release fromthe gel received at various time periods and incubated thecells for 24 hours The results of our experiments revealed(Figure 2(b)) that higher concentrations of RA (025ndash05mgmL) protected the cells from the damage caused bydifferent concentrations of H

2O2and enhanced cell viability

by 10ndash24 Moreover 05mgmL of RA restored HaCaT cellviability until the control level in the presence of 100120583M ofH2O2

It is possible that the protective effect of RA is relatedto its antioxidant activity Oxidative stress conditions in cellsdevelop in the presence of an imbalance between ROS gen-eration and endogenous antioxidant defense mechanismsThis state of oxidative stress can affect all important cellularcomponents like proteins DNA andmembrane lipids whichis considered to be the main mechanism of cell damage[30] There is research confirming that exogenous moleculesfrom dietary sources such as polyphenols are very efficientin preventing the alteration caused by oxidative stress [31]because they scavenge and suppress the formation of free rad-icals Thus the use of preparations with antioxidant activity iscritical for cells that are in oxidative stress conditions

34 The Antioxidant Activity of Rosmarinic Acid in NormalConditions and under Oxidative Stress In order to evaluatethe antioxidant activity of different concentrations of RAin normal conditions and under oxidative stress in thenext series of experiments we measured the quantity ofthe intracellular ROS in cells using the DCFH-DA dye(Figures 3(a) and 3(b)) For these experiments we choseH2O2concentration of 100120583M because this is the lowest

concentration which after 24 hours statically significantlyreduces cell viability It is known that H

2O2should relatively

easily diffuse intoout of the cell through lipid bilayer oraquaporins In our experiments (Figure 3(b)) this was clearlyvisible since the amount of intracellular ROS in cells thatwere affected by 100 120583M of H

2O2during the original period

(05 h) increased by 30 whereas in control cells (withoutH2O2) the amount of ROS increased by an average of 7ndash

10 The lowest amount of RA (005mgmL) that affectedkeratinocyte cells only slightly reduced the amount of ROS(by 43) but all the higher amounts of RA that were used in

Evidence-Based Complementary and Alternative Medicine 5

Cel

l via

bilit

y (

)120

100

80

60

40

20

0Control 50 100 150 200 300 400 500

lowast

lowast

lowast

lowast

lowast

lowast

(120583M H2O2)

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0

Control

100 150 200

lowast

lowast

lowast

lowast

lowast

lowast

H2O2RA 005mg and H2O2

RA 025mg and H2O2RA 05mg and H2O2

(120583M H2O2)

(b)

Figure 2 Effect of different concentrations ofH2O2andRAonHaCaT cell viability HaCaT cells were treatedwith (a) different concentrations

(50ndash500 120583M) of H2O2for 24 hours and (b) different concentrations (005ndash05mgmL) of RA and H

2O2(100ndash200 120583M) for 24 hours Cell

viability was assessed using the MTT method Data are presented as means of the percentage of the untreated control cells plusmn SE (119899 = 4)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

4

2

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

(a)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

Control + 100120583M H2O2

(b)

Figure 3 Effects of different concentrations of rosmarinic acid on intracellular ROS generation in HaCaT cells Cells were preincubated withDCFH-DA (10 120583M) for 30min and then washed twice and (a) treated with different concentrations of RA and (b) treated with 100120583MH

2O2

and different concentrations of RA Control cells were treated with the same amounts of the solvent The number of experiments is 3

the study in all the studied time periods significantly reducedintracellular ROS quantity After 3 hours the amount of ROSwas below 14ndash28 at 01ndash05mgmL of RA In cells affected by05mg of RA the amount of ROS was close to that observedin the control cells

The skin is the interface between the body and itsenvironment and acts as a barrier protecting from adverseexternal factors Keratinocytes are the principal cell typecomprising the epidermis and constituting 90 of the total

amount of epidermal cells [32] For this reason the humankeratinocyte cell line HaCaT that was used in this researchwas an excellent model to evaluate the biological effect ofthe active substance of themodeled preparations Chemicallyrosmarinic acid is a derivative of two connected phenolicacids (coffee and 34-dihydroxyphenyl lactic acid) and has4 OH groups There are studies showing that it is the mostpotent antioxidant among the hydroxycinnamic acids [33]Studies have shown that rosmarinic acid is an effective

6 Evidence-Based Complementary and Alternative Medicine

scavenger of the DPPH radical and it also scavenges thereactive nitrogen species peroxynitrite and various ROS[34] The results of experiments done with cell cultures showthat through its antioxidant activity RA could be able toattenuate H

2O2-induced cell injury [35] The results of our

experiments on skin cells also demonstrated that RA haddose-dependent antioxidant activity In normal conditions05mgmL of RA after 3 hours reduced the amount ofintracellular ROS to 23 (Figure 3(a)) Therefore in normalconditions the examined preparations can provide protectiveand antiaging effects Scientific data confirmed that manyexternal factors (such as UV light traumas ultrasoundinfections air pollutants and cigarette smoke) and internalfactors (drugs contaminated food and some diseases suchas atopic dermatitis) cause increased ROS levels in skin cellsMost of the ROS have a short life span and are immobile (eghydroxyl or superoxide radicals) and they immediately (atthe source) react with the surrounding biomolecules H

2O2

is stable and easily diffusible in an aqueous environment Iteasily passes through biological membranes and therefore issuitable to create oxidative stress in experimental conditionsCell viability-decreasing H

2O2concentrations determined in

our experiments with HaCaT cells (EC501837 plusmn 204) were

similar to those found by Liu et al [22] Investigations of otherauthors demonstrate that the first signs of cell impairmentare noted at 50 120583M H

2O2 On the other hand 150 120583M of

H2O2and 300 120583MofH

2O2are relatively high doses to induce

apoptosis in Jurkat T cells and in HaCaT cells respectively[32 36] It was shown that very severe oxidative stressactivates a large number of signaling pathways in cells andcauses cell death via either apoptotic or necrotic mechanisms[37] The RA concentration of 005mgmL slightly reducedthe amount of intracellular ROS and had only limited effecton the oxidative state of cells Higher amounts of RA (01and 025mgmL) decreased the level of ROS by 14 and 22respectively However the amount of radicals in cells wasstill higher than that in the controls In normal conditionsskin cells do not sustain that much damage from harmfulenvironmental factors to initiate the production of such largeamounts of ROS that they would cause cell death Only with05mgmL of RA a decrease in the amount of ROS by 28was achieved and the remaining amount of ROS was similarto that in the control cells Based on these findings it is clearthat preparations with lemon balm extract can reduce theamount of ROS increased because of unfavorable externalconditions the effect of internal factors on the body or apathologic skin process in intracellular keratinocytes

4 Conclusions

Lemon balm hydrogels were modeled using carbomermethylcellulose and mixtures of them as gelling materialsThe study showed that the used gelling material affected thephysicochemical properties of semisolid preparations Theresults of biopharmaceutical tests in vitro showed that eventhough the highest amount of released RA was found in themethylcellulose-based gels carbomer (up to 10) due to itssemisolid consistency was a more appropriate base for theinsertion of lemon balm extracts The results of biological

investigations showed that rosmarinic acid depending on theconcentration reduced the amount of intracellular ROS inhuman keratinocyte cells and enhanced cell viability underoxidative stress

Conflict of Interests

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

References

[1] R Vejdani H R M Shalmani M Mir-Fattahi et al ldquoTheefficacy of an herbal medicine Carmint on the relief ofabdominal pain and bloating in patients with irritable bowelsyndrome a pilot studyrdquoDigestive Diseases and Sciences vol 51no 8 pp 1501ndash1507 2006

[2] J Lee E Jung J Koh Y S Kim and D Park ldquoEffect ofrosmarinic acid on atopic dermatitisrdquo Journal of Dermatologyvol 35 no 12 pp 768ndash771 2008

[3] E N Martins N T C Pessano L Leal et al ldquoProtectiveeffect of Melissa officinalis aqueous extract against Mn-inducedoxidative stress in chronically exposed micerdquo Brain ResearchBulletin vol 87 no 1 pp 74ndash79 2012

[4] H Moradkhani E Sargsyan H Bibak et al ldquoMelissa officinalisL a valuable medicine plant a reviewrdquo Journal of MedicinalPlants Research vol 4 no 25 pp 2753ndash2759 2010

[5] A Zarei S Changizi-Ashtiyani S Taheri and N Hosseini ldquoAbrief overviewof the effects ofMelissa officinalisL extract on thefunction of various body organsrdquo Zahedan Journal of Researchin Medical Sciences vol 15 pp 29ndash34 2015

[6] A Arceusz andMWesolowski ldquoQuality consistency evaluationof Melissa officinalis L commercial herbs by HPLC fingerprintand quantitation of selected phenolic acidsrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 83 pp 215ndash220 2013

[7] A Astani J Reichling and P Schnitzler ldquoMelissa officinalisextract inhibits attachment of herpes simplex virus in vitrordquoChemotherapy vol 58 no 1 pp 70ndash77 2012

[8] R Awad A Muhammad T Durst V L Trudeau and JT Arnason ldquoBioassay-guided fractionation of lemon balm(Melissa officinalis L) using an in vitro measure of GABAtransaminase activityrdquo Phytotherapy Research vol 23 no 8 pp1075ndash1081 2009

[9] A E Ince S Sahin and S G Sumnu ldquoExtraction of phenoliccompounds from melissa using microwave and ultrasoundrdquoTurkish Journal of Agriculture and Forestry vol 37 no 1 pp 69ndash75 2013

[10] G Mazzanti L Battinelli C Pompeo et al ldquoInhibitory activityof Melissa officinalis L extract on Herpes simplex virus type 2replicationrdquo Natural Product Research vol 22 no 16 pp 1433ndash1440 2008

[11] MMorrison LemonBalm httpwwwherballegacycomMor-rison Lemon Balmhtml

[12] A Bounihi GHajjaj R Alnamer Y Cherrah andA Zellou ldquoInvivo potential anti-inflammatory activity of Melissa officinalisL essential oilrdquoAdvances in Pharmacological Sciences vol 2013Article ID 101759 7 pages 2013

[13] M A Ribeiro M G Bernardo-Gil and M M EsquıvelldquoMelissa officinalis L study of antioxidant activity in supercrit-ical residuesrdquo Journal of Supercritical Fluids vol 21 no 1 pp51ndash60 2001

Evidence-Based Complementary and Alternative Medicine 7

[14] M Foldvari ldquoNon-invasive administration of drugs throughthe skin challenges in delivery system designrdquo PharmaceuticalScience amp Technology Today vol 3 no 12 pp 417ndash425 2000

[15] AVerma S Singh R Kaur A Kumar andUK Jain ldquoFormula-tion optimization and evaluation of clobetasol propionate gelrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 666ndash674 2013

[16] M G Dodov K Goracinova M Simonoska S Trajkovic-Jolevska J T Ribarska and M D Mitevska ldquoFormulation andevaluation of diazepamhydrogel for rectal administrationrdquoActaPharmaceutica vol 55 no 3 pp 251ndash261 2005

[17] FDAGuidance for Industry Nonsterile Semisolid Dosage FormsScale-Up and Postapproval Changes Chemistry Manufacturingand Controls In Vitro Release Testing and In Vivo BioequivalenceDocumentation FDA 1997

[18] G L Flynn V P Shah S N Tenjarla et al ldquoAssessment of valueand applications of in vitro testing of topical dermatologicaldrug productsrdquoPharmaceutical Research vol 16 no 9 pp 1325ndash1330 1999

[19] A Stelmakiene K Ramanauskiene and V Briedis ldquoRelease ofrosmarinic acid from semisolid formulations and its penetra-tion through human skin ex vivordquo Acta Pharmaceutica vol 65no 2 pp 199ndash205 2015

[20] M Zilius K Ramanauskiene and V Briedis ldquoRelease ofpropolis phenolic acids from semisolid formulations and theirpenetration into the human skin in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID958717 7 pages 2013

[21] F J Ahmad M A Alam Z I Khan R K Khar and MAli ldquoDevelopment and in vitro evaluation of an acid bufferingbioadhesive vaginal gel for mixed vaginal infectionsrdquo ActaPharmaceutica vol 58 no 4 pp 407ndash419 2008

[22] L Liu H Xie X Chen et al ldquoDifferential response of normalhuman epidermal keratinocytes and HaCaT cells to hydrogenperoxide-induced oxidative stressrdquo Clinical amp ExperimentalDermatology vol 37 no 7 pp 772ndash780 2012

[23] V Armoskaite K Ramanauskiene and V Briedis ldquoEvaluationof base for optimal drug delivery for iontophoretic therapyinvestigation of quality and stabilityrdquo African Journal of Phar-macy and Pharmacology vol 6 pp 1685ndash1695 2012

[24] M-H Schmid-Wendtner and H C Korting ldquoThe pH of theskin surface and its impact on the barrier functionrdquo SkinPharmacology and Physiology vol 19 no 6 pp 296ndash302 2006

[25] K Rizi R J Green M X Donaldson and A C WilliamsldquoUsing pH abnormalities in diseased skin to trigger and targettopical therapyrdquo Pharmaceutical Research vol 28 no 10 pp2589ndash2598 2011

[26] K Cal ldquoAcross skin barrier known methods new perfor-mancesrdquo Frontiers in Drug Design amp Discovery vol 4 no 1 pp162ndash188 2009

[27] D R Drake K A Brogden D V Dawson and P W WertzldquoThematic review series skin lipids Antimicrobial lipids at theskin surfacerdquo The Journal of Lipid Research vol 49 no 1 pp4ndash11 2008

[28] G P Moss J C Dearden H Patel and M T D CroninldquoQuantitative structure-permeability relationships (QSPRs) forpercutaneous absorptionrdquo Toxicology in Vitro vol 16 no 3 pp299ndash317 2002

[29] D Touboul F Halgand A Brunelle et al ldquoTissue molecularion imaging by gold cluster ion bombardmentrdquo AnalyticalChemistry vol 76 no 6 pp 1550ndash1559 2004

[30] N Sivaranjani S V Rao andG Rajeev ldquoRole of reactive oxygenspecies and antioxidants in atopic dermatitisrdquo Journal of Clinicaland Diagnostic Research vol 7 no 12 pp 2683ndash2685 2013

[31] O Fadel K El Kirat and S Morandat ldquoThe natural antioxidantrosmarinic acid spontaneously penetratesmembranes to inhibitlipid peroxidation in siturdquo Biochimica et Biophysica ActamdashBiomembranes vol 1808 no 12 pp 2973ndash2980 2011

[32] E-R Lee Y-J Kang J-H KimH T Lee and S-G Cho ldquoMod-ulation of apoptosis in HaCaT keratinocytes via differentialregulation of ERK signaling pathway by flavonoidsrdquoThe Journalof Biological Chemistry vol 280 no 36 pp 31498ndash31507 2005

[33] M A Soobrattee V S Neergheen A Luximon-Ramma OI Aruoma and T Bahorun ldquoPhenolics as potential antiox-idant therapeutic agents Mechanism and actionsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

[34] NAKelseyHMWilkins andDA Linseman ldquoNutraceuticalantioxidants as novel neuroprotective agentsrdquoMolecules vol 15no 11 pp 7792ndash7814 2010

[35] L P Gao H L Wei H S Zhao S Y Xiao and R-L ZhengldquoAntiapoptotic and antioxidant effects of rosmarinic acid inastrocytesrdquo Pharmazie vol 60 no 1 pp 62ndash65 2005

[36] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[37] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Cel

l via

bilit

y (

)120

100

80

60

40

20

0Control 50 100 150 200 300 400 500

lowast

lowast

lowast

lowast

lowast

lowast

(120583M H2O2)

(a)

Cel

l via

bilit

y (

)

120

100

80

60

40

20

0

Control

100 150 200

lowast

lowast

lowast

lowast

lowast

lowast

H2O2RA 005mg and H2O2

RA 025mg and H2O2RA 05mg and H2O2

(120583M H2O2)

(b)

Figure 2 Effect of different concentrations ofH2O2andRAonHaCaT cell viability HaCaT cells were treatedwith (a) different concentrations

(50ndash500 120583M) of H2O2for 24 hours and (b) different concentrations (005ndash05mgmL) of RA and H

2O2(100ndash200 120583M) for 24 hours Cell

viability was assessed using the MTT method Data are presented as means of the percentage of the untreated control cells plusmn SE (119899 = 4)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

4

2

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

(a)

Fluo

resc

ence

inte

nsiv

ity

14

12

10

8

6

Time (h)00 05 10 15 20 25 30

Control005mg RA01mg RA

025mg RA05mg RA

Control + 100120583M H2O2

(b)

Figure 3 Effects of different concentrations of rosmarinic acid on intracellular ROS generation in HaCaT cells Cells were preincubated withDCFH-DA (10 120583M) for 30min and then washed twice and (a) treated with different concentrations of RA and (b) treated with 100120583MH

2O2

and different concentrations of RA Control cells were treated with the same amounts of the solvent The number of experiments is 3

the study in all the studied time periods significantly reducedintracellular ROS quantity After 3 hours the amount of ROSwas below 14ndash28 at 01ndash05mgmL of RA In cells affected by05mg of RA the amount of ROS was close to that observedin the control cells

The skin is the interface between the body and itsenvironment and acts as a barrier protecting from adverseexternal factors Keratinocytes are the principal cell typecomprising the epidermis and constituting 90 of the total

amount of epidermal cells [32] For this reason the humankeratinocyte cell line HaCaT that was used in this researchwas an excellent model to evaluate the biological effect ofthe active substance of themodeled preparations Chemicallyrosmarinic acid is a derivative of two connected phenolicacids (coffee and 34-dihydroxyphenyl lactic acid) and has4 OH groups There are studies showing that it is the mostpotent antioxidant among the hydroxycinnamic acids [33]Studies have shown that rosmarinic acid is an effective

6 Evidence-Based Complementary and Alternative Medicine

scavenger of the DPPH radical and it also scavenges thereactive nitrogen species peroxynitrite and various ROS[34] The results of experiments done with cell cultures showthat through its antioxidant activity RA could be able toattenuate H

2O2-induced cell injury [35] The results of our

experiments on skin cells also demonstrated that RA haddose-dependent antioxidant activity In normal conditions05mgmL of RA after 3 hours reduced the amount ofintracellular ROS to 23 (Figure 3(a)) Therefore in normalconditions the examined preparations can provide protectiveand antiaging effects Scientific data confirmed that manyexternal factors (such as UV light traumas ultrasoundinfections air pollutants and cigarette smoke) and internalfactors (drugs contaminated food and some diseases suchas atopic dermatitis) cause increased ROS levels in skin cellsMost of the ROS have a short life span and are immobile (eghydroxyl or superoxide radicals) and they immediately (atthe source) react with the surrounding biomolecules H

2O2

is stable and easily diffusible in an aqueous environment Iteasily passes through biological membranes and therefore issuitable to create oxidative stress in experimental conditionsCell viability-decreasing H

2O2concentrations determined in

our experiments with HaCaT cells (EC501837 plusmn 204) were

similar to those found by Liu et al [22] Investigations of otherauthors demonstrate that the first signs of cell impairmentare noted at 50 120583M H

2O2 On the other hand 150 120583M of

H2O2and 300 120583MofH

2O2are relatively high doses to induce

apoptosis in Jurkat T cells and in HaCaT cells respectively[32 36] It was shown that very severe oxidative stressactivates a large number of signaling pathways in cells andcauses cell death via either apoptotic or necrotic mechanisms[37] The RA concentration of 005mgmL slightly reducedthe amount of intracellular ROS and had only limited effecton the oxidative state of cells Higher amounts of RA (01and 025mgmL) decreased the level of ROS by 14 and 22respectively However the amount of radicals in cells wasstill higher than that in the controls In normal conditionsskin cells do not sustain that much damage from harmfulenvironmental factors to initiate the production of such largeamounts of ROS that they would cause cell death Only with05mgmL of RA a decrease in the amount of ROS by 28was achieved and the remaining amount of ROS was similarto that in the control cells Based on these findings it is clearthat preparations with lemon balm extract can reduce theamount of ROS increased because of unfavorable externalconditions the effect of internal factors on the body or apathologic skin process in intracellular keratinocytes

4 Conclusions

Lemon balm hydrogels were modeled using carbomermethylcellulose and mixtures of them as gelling materialsThe study showed that the used gelling material affected thephysicochemical properties of semisolid preparations Theresults of biopharmaceutical tests in vitro showed that eventhough the highest amount of released RA was found in themethylcellulose-based gels carbomer (up to 10) due to itssemisolid consistency was a more appropriate base for theinsertion of lemon balm extracts The results of biological

investigations showed that rosmarinic acid depending on theconcentration reduced the amount of intracellular ROS inhuman keratinocyte cells and enhanced cell viability underoxidative stress

Conflict of Interests

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

References

[1] R Vejdani H R M Shalmani M Mir-Fattahi et al ldquoTheefficacy of an herbal medicine Carmint on the relief ofabdominal pain and bloating in patients with irritable bowelsyndrome a pilot studyrdquoDigestive Diseases and Sciences vol 51no 8 pp 1501ndash1507 2006

[2] J Lee E Jung J Koh Y S Kim and D Park ldquoEffect ofrosmarinic acid on atopic dermatitisrdquo Journal of Dermatologyvol 35 no 12 pp 768ndash771 2008

[3] E N Martins N T C Pessano L Leal et al ldquoProtectiveeffect of Melissa officinalis aqueous extract against Mn-inducedoxidative stress in chronically exposed micerdquo Brain ResearchBulletin vol 87 no 1 pp 74ndash79 2012

[4] H Moradkhani E Sargsyan H Bibak et al ldquoMelissa officinalisL a valuable medicine plant a reviewrdquo Journal of MedicinalPlants Research vol 4 no 25 pp 2753ndash2759 2010

[5] A Zarei S Changizi-Ashtiyani S Taheri and N Hosseini ldquoAbrief overviewof the effects ofMelissa officinalisL extract on thefunction of various body organsrdquo Zahedan Journal of Researchin Medical Sciences vol 15 pp 29ndash34 2015

[6] A Arceusz andMWesolowski ldquoQuality consistency evaluationof Melissa officinalis L commercial herbs by HPLC fingerprintand quantitation of selected phenolic acidsrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 83 pp 215ndash220 2013

[7] A Astani J Reichling and P Schnitzler ldquoMelissa officinalisextract inhibits attachment of herpes simplex virus in vitrordquoChemotherapy vol 58 no 1 pp 70ndash77 2012

[8] R Awad A Muhammad T Durst V L Trudeau and JT Arnason ldquoBioassay-guided fractionation of lemon balm(Melissa officinalis L) using an in vitro measure of GABAtransaminase activityrdquo Phytotherapy Research vol 23 no 8 pp1075ndash1081 2009

[9] A E Ince S Sahin and S G Sumnu ldquoExtraction of phenoliccompounds from melissa using microwave and ultrasoundrdquoTurkish Journal of Agriculture and Forestry vol 37 no 1 pp 69ndash75 2013

[10] G Mazzanti L Battinelli C Pompeo et al ldquoInhibitory activityof Melissa officinalis L extract on Herpes simplex virus type 2replicationrdquo Natural Product Research vol 22 no 16 pp 1433ndash1440 2008

[11] MMorrison LemonBalm httpwwwherballegacycomMor-rison Lemon Balmhtml

[12] A Bounihi GHajjaj R Alnamer Y Cherrah andA Zellou ldquoInvivo potential anti-inflammatory activity of Melissa officinalisL essential oilrdquoAdvances in Pharmacological Sciences vol 2013Article ID 101759 7 pages 2013

[13] M A Ribeiro M G Bernardo-Gil and M M EsquıvelldquoMelissa officinalis L study of antioxidant activity in supercrit-ical residuesrdquo Journal of Supercritical Fluids vol 21 no 1 pp51ndash60 2001

Evidence-Based Complementary and Alternative Medicine 7

[14] M Foldvari ldquoNon-invasive administration of drugs throughthe skin challenges in delivery system designrdquo PharmaceuticalScience amp Technology Today vol 3 no 12 pp 417ndash425 2000

[15] AVerma S Singh R Kaur A Kumar andUK Jain ldquoFormula-tion optimization and evaluation of clobetasol propionate gelrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 666ndash674 2013

[16] M G Dodov K Goracinova M Simonoska S Trajkovic-Jolevska J T Ribarska and M D Mitevska ldquoFormulation andevaluation of diazepamhydrogel for rectal administrationrdquoActaPharmaceutica vol 55 no 3 pp 251ndash261 2005

[17] FDAGuidance for Industry Nonsterile Semisolid Dosage FormsScale-Up and Postapproval Changes Chemistry Manufacturingand Controls In Vitro Release Testing and In Vivo BioequivalenceDocumentation FDA 1997

[18] G L Flynn V P Shah S N Tenjarla et al ldquoAssessment of valueand applications of in vitro testing of topical dermatologicaldrug productsrdquoPharmaceutical Research vol 16 no 9 pp 1325ndash1330 1999

[19] A Stelmakiene K Ramanauskiene and V Briedis ldquoRelease ofrosmarinic acid from semisolid formulations and its penetra-tion through human skin ex vivordquo Acta Pharmaceutica vol 65no 2 pp 199ndash205 2015

[20] M Zilius K Ramanauskiene and V Briedis ldquoRelease ofpropolis phenolic acids from semisolid formulations and theirpenetration into the human skin in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID958717 7 pages 2013

[21] F J Ahmad M A Alam Z I Khan R K Khar and MAli ldquoDevelopment and in vitro evaluation of an acid bufferingbioadhesive vaginal gel for mixed vaginal infectionsrdquo ActaPharmaceutica vol 58 no 4 pp 407ndash419 2008

[22] L Liu H Xie X Chen et al ldquoDifferential response of normalhuman epidermal keratinocytes and HaCaT cells to hydrogenperoxide-induced oxidative stressrdquo Clinical amp ExperimentalDermatology vol 37 no 7 pp 772ndash780 2012

[23] V Armoskaite K Ramanauskiene and V Briedis ldquoEvaluationof base for optimal drug delivery for iontophoretic therapyinvestigation of quality and stabilityrdquo African Journal of Phar-macy and Pharmacology vol 6 pp 1685ndash1695 2012

[24] M-H Schmid-Wendtner and H C Korting ldquoThe pH of theskin surface and its impact on the barrier functionrdquo SkinPharmacology and Physiology vol 19 no 6 pp 296ndash302 2006

[25] K Rizi R J Green M X Donaldson and A C WilliamsldquoUsing pH abnormalities in diseased skin to trigger and targettopical therapyrdquo Pharmaceutical Research vol 28 no 10 pp2589ndash2598 2011

[26] K Cal ldquoAcross skin barrier known methods new perfor-mancesrdquo Frontiers in Drug Design amp Discovery vol 4 no 1 pp162ndash188 2009

[27] D R Drake K A Brogden D V Dawson and P W WertzldquoThematic review series skin lipids Antimicrobial lipids at theskin surfacerdquo The Journal of Lipid Research vol 49 no 1 pp4ndash11 2008

[28] G P Moss J C Dearden H Patel and M T D CroninldquoQuantitative structure-permeability relationships (QSPRs) forpercutaneous absorptionrdquo Toxicology in Vitro vol 16 no 3 pp299ndash317 2002

[29] D Touboul F Halgand A Brunelle et al ldquoTissue molecularion imaging by gold cluster ion bombardmentrdquo AnalyticalChemistry vol 76 no 6 pp 1550ndash1559 2004

[30] N Sivaranjani S V Rao andG Rajeev ldquoRole of reactive oxygenspecies and antioxidants in atopic dermatitisrdquo Journal of Clinicaland Diagnostic Research vol 7 no 12 pp 2683ndash2685 2013

[31] O Fadel K El Kirat and S Morandat ldquoThe natural antioxidantrosmarinic acid spontaneously penetratesmembranes to inhibitlipid peroxidation in siturdquo Biochimica et Biophysica ActamdashBiomembranes vol 1808 no 12 pp 2973ndash2980 2011

[32] E-R Lee Y-J Kang J-H KimH T Lee and S-G Cho ldquoMod-ulation of apoptosis in HaCaT keratinocytes via differentialregulation of ERK signaling pathway by flavonoidsrdquoThe Journalof Biological Chemistry vol 280 no 36 pp 31498ndash31507 2005

[33] M A Soobrattee V S Neergheen A Luximon-Ramma OI Aruoma and T Bahorun ldquoPhenolics as potential antiox-idant therapeutic agents Mechanism and actionsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

[34] NAKelseyHMWilkins andDA Linseman ldquoNutraceuticalantioxidants as novel neuroprotective agentsrdquoMolecules vol 15no 11 pp 7792ndash7814 2010

[35] L P Gao H L Wei H S Zhao S Y Xiao and R-L ZhengldquoAntiapoptotic and antioxidant effects of rosmarinic acid inastrocytesrdquo Pharmazie vol 60 no 1 pp 62ndash65 2005

[36] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[37] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

scavenger of the DPPH radical and it also scavenges thereactive nitrogen species peroxynitrite and various ROS[34] The results of experiments done with cell cultures showthat through its antioxidant activity RA could be able toattenuate H

2O2-induced cell injury [35] The results of our

experiments on skin cells also demonstrated that RA haddose-dependent antioxidant activity In normal conditions05mgmL of RA after 3 hours reduced the amount ofintracellular ROS to 23 (Figure 3(a)) Therefore in normalconditions the examined preparations can provide protectiveand antiaging effects Scientific data confirmed that manyexternal factors (such as UV light traumas ultrasoundinfections air pollutants and cigarette smoke) and internalfactors (drugs contaminated food and some diseases suchas atopic dermatitis) cause increased ROS levels in skin cellsMost of the ROS have a short life span and are immobile (eghydroxyl or superoxide radicals) and they immediately (atthe source) react with the surrounding biomolecules H

2O2

is stable and easily diffusible in an aqueous environment Iteasily passes through biological membranes and therefore issuitable to create oxidative stress in experimental conditionsCell viability-decreasing H

2O2concentrations determined in

our experiments with HaCaT cells (EC501837 plusmn 204) were

similar to those found by Liu et al [22] Investigations of otherauthors demonstrate that the first signs of cell impairmentare noted at 50 120583M H

2O2 On the other hand 150 120583M of

H2O2and 300 120583MofH

2O2are relatively high doses to induce

apoptosis in Jurkat T cells and in HaCaT cells respectively[32 36] It was shown that very severe oxidative stressactivates a large number of signaling pathways in cells andcauses cell death via either apoptotic or necrotic mechanisms[37] The RA concentration of 005mgmL slightly reducedthe amount of intracellular ROS and had only limited effecton the oxidative state of cells Higher amounts of RA (01and 025mgmL) decreased the level of ROS by 14 and 22respectively However the amount of radicals in cells wasstill higher than that in the controls In normal conditionsskin cells do not sustain that much damage from harmfulenvironmental factors to initiate the production of such largeamounts of ROS that they would cause cell death Only with05mgmL of RA a decrease in the amount of ROS by 28was achieved and the remaining amount of ROS was similarto that in the control cells Based on these findings it is clearthat preparations with lemon balm extract can reduce theamount of ROS increased because of unfavorable externalconditions the effect of internal factors on the body or apathologic skin process in intracellular keratinocytes

4 Conclusions

Lemon balm hydrogels were modeled using carbomermethylcellulose and mixtures of them as gelling materialsThe study showed that the used gelling material affected thephysicochemical properties of semisolid preparations Theresults of biopharmaceutical tests in vitro showed that eventhough the highest amount of released RA was found in themethylcellulose-based gels carbomer (up to 10) due to itssemisolid consistency was a more appropriate base for theinsertion of lemon balm extracts The results of biological

investigations showed that rosmarinic acid depending on theconcentration reduced the amount of intracellular ROS inhuman keratinocyte cells and enhanced cell viability underoxidative stress

Conflict of Interests

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

References

[1] R Vejdani H R M Shalmani M Mir-Fattahi et al ldquoTheefficacy of an herbal medicine Carmint on the relief ofabdominal pain and bloating in patients with irritable bowelsyndrome a pilot studyrdquoDigestive Diseases and Sciences vol 51no 8 pp 1501ndash1507 2006

[2] J Lee E Jung J Koh Y S Kim and D Park ldquoEffect ofrosmarinic acid on atopic dermatitisrdquo Journal of Dermatologyvol 35 no 12 pp 768ndash771 2008

[3] E N Martins N T C Pessano L Leal et al ldquoProtectiveeffect of Melissa officinalis aqueous extract against Mn-inducedoxidative stress in chronically exposed micerdquo Brain ResearchBulletin vol 87 no 1 pp 74ndash79 2012

[4] H Moradkhani E Sargsyan H Bibak et al ldquoMelissa officinalisL a valuable medicine plant a reviewrdquo Journal of MedicinalPlants Research vol 4 no 25 pp 2753ndash2759 2010

[5] A Zarei S Changizi-Ashtiyani S Taheri and N Hosseini ldquoAbrief overviewof the effects ofMelissa officinalisL extract on thefunction of various body organsrdquo Zahedan Journal of Researchin Medical Sciences vol 15 pp 29ndash34 2015

[6] A Arceusz andMWesolowski ldquoQuality consistency evaluationof Melissa officinalis L commercial herbs by HPLC fingerprintand quantitation of selected phenolic acidsrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 83 pp 215ndash220 2013

[7] A Astani J Reichling and P Schnitzler ldquoMelissa officinalisextract inhibits attachment of herpes simplex virus in vitrordquoChemotherapy vol 58 no 1 pp 70ndash77 2012

[8] R Awad A Muhammad T Durst V L Trudeau and JT Arnason ldquoBioassay-guided fractionation of lemon balm(Melissa officinalis L) using an in vitro measure of GABAtransaminase activityrdquo Phytotherapy Research vol 23 no 8 pp1075ndash1081 2009

[9] A E Ince S Sahin and S G Sumnu ldquoExtraction of phenoliccompounds from melissa using microwave and ultrasoundrdquoTurkish Journal of Agriculture and Forestry vol 37 no 1 pp 69ndash75 2013

[10] G Mazzanti L Battinelli C Pompeo et al ldquoInhibitory activityof Melissa officinalis L extract on Herpes simplex virus type 2replicationrdquo Natural Product Research vol 22 no 16 pp 1433ndash1440 2008

[11] MMorrison LemonBalm httpwwwherballegacycomMor-rison Lemon Balmhtml

[12] A Bounihi GHajjaj R Alnamer Y Cherrah andA Zellou ldquoInvivo potential anti-inflammatory activity of Melissa officinalisL essential oilrdquoAdvances in Pharmacological Sciences vol 2013Article ID 101759 7 pages 2013

[13] M A Ribeiro M G Bernardo-Gil and M M EsquıvelldquoMelissa officinalis L study of antioxidant activity in supercrit-ical residuesrdquo Journal of Supercritical Fluids vol 21 no 1 pp51ndash60 2001

Evidence-Based Complementary and Alternative Medicine 7

[14] M Foldvari ldquoNon-invasive administration of drugs throughthe skin challenges in delivery system designrdquo PharmaceuticalScience amp Technology Today vol 3 no 12 pp 417ndash425 2000

[15] AVerma S Singh R Kaur A Kumar andUK Jain ldquoFormula-tion optimization and evaluation of clobetasol propionate gelrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 666ndash674 2013

[16] M G Dodov K Goracinova M Simonoska S Trajkovic-Jolevska J T Ribarska and M D Mitevska ldquoFormulation andevaluation of diazepamhydrogel for rectal administrationrdquoActaPharmaceutica vol 55 no 3 pp 251ndash261 2005

[17] FDAGuidance for Industry Nonsterile Semisolid Dosage FormsScale-Up and Postapproval Changes Chemistry Manufacturingand Controls In Vitro Release Testing and In Vivo BioequivalenceDocumentation FDA 1997

[18] G L Flynn V P Shah S N Tenjarla et al ldquoAssessment of valueand applications of in vitro testing of topical dermatologicaldrug productsrdquoPharmaceutical Research vol 16 no 9 pp 1325ndash1330 1999

[19] A Stelmakiene K Ramanauskiene and V Briedis ldquoRelease ofrosmarinic acid from semisolid formulations and its penetra-tion through human skin ex vivordquo Acta Pharmaceutica vol 65no 2 pp 199ndash205 2015

[20] M Zilius K Ramanauskiene and V Briedis ldquoRelease ofpropolis phenolic acids from semisolid formulations and theirpenetration into the human skin in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID958717 7 pages 2013

[21] F J Ahmad M A Alam Z I Khan R K Khar and MAli ldquoDevelopment and in vitro evaluation of an acid bufferingbioadhesive vaginal gel for mixed vaginal infectionsrdquo ActaPharmaceutica vol 58 no 4 pp 407ndash419 2008

[22] L Liu H Xie X Chen et al ldquoDifferential response of normalhuman epidermal keratinocytes and HaCaT cells to hydrogenperoxide-induced oxidative stressrdquo Clinical amp ExperimentalDermatology vol 37 no 7 pp 772ndash780 2012

[23] V Armoskaite K Ramanauskiene and V Briedis ldquoEvaluationof base for optimal drug delivery for iontophoretic therapyinvestigation of quality and stabilityrdquo African Journal of Phar-macy and Pharmacology vol 6 pp 1685ndash1695 2012

[24] M-H Schmid-Wendtner and H C Korting ldquoThe pH of theskin surface and its impact on the barrier functionrdquo SkinPharmacology and Physiology vol 19 no 6 pp 296ndash302 2006

[25] K Rizi R J Green M X Donaldson and A C WilliamsldquoUsing pH abnormalities in diseased skin to trigger and targettopical therapyrdquo Pharmaceutical Research vol 28 no 10 pp2589ndash2598 2011

[26] K Cal ldquoAcross skin barrier known methods new perfor-mancesrdquo Frontiers in Drug Design amp Discovery vol 4 no 1 pp162ndash188 2009

[27] D R Drake K A Brogden D V Dawson and P W WertzldquoThematic review series skin lipids Antimicrobial lipids at theskin surfacerdquo The Journal of Lipid Research vol 49 no 1 pp4ndash11 2008

[28] G P Moss J C Dearden H Patel and M T D CroninldquoQuantitative structure-permeability relationships (QSPRs) forpercutaneous absorptionrdquo Toxicology in Vitro vol 16 no 3 pp299ndash317 2002

[29] D Touboul F Halgand A Brunelle et al ldquoTissue molecularion imaging by gold cluster ion bombardmentrdquo AnalyticalChemistry vol 76 no 6 pp 1550ndash1559 2004

[30] N Sivaranjani S V Rao andG Rajeev ldquoRole of reactive oxygenspecies and antioxidants in atopic dermatitisrdquo Journal of Clinicaland Diagnostic Research vol 7 no 12 pp 2683ndash2685 2013

[31] O Fadel K El Kirat and S Morandat ldquoThe natural antioxidantrosmarinic acid spontaneously penetratesmembranes to inhibitlipid peroxidation in siturdquo Biochimica et Biophysica ActamdashBiomembranes vol 1808 no 12 pp 2973ndash2980 2011

[32] E-R Lee Y-J Kang J-H KimH T Lee and S-G Cho ldquoMod-ulation of apoptosis in HaCaT keratinocytes via differentialregulation of ERK signaling pathway by flavonoidsrdquoThe Journalof Biological Chemistry vol 280 no 36 pp 31498ndash31507 2005

[33] M A Soobrattee V S Neergheen A Luximon-Ramma OI Aruoma and T Bahorun ldquoPhenolics as potential antiox-idant therapeutic agents Mechanism and actionsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

[34] NAKelseyHMWilkins andDA Linseman ldquoNutraceuticalantioxidants as novel neuroprotective agentsrdquoMolecules vol 15no 11 pp 7792ndash7814 2010

[35] L P Gao H L Wei H S Zhao S Y Xiao and R-L ZhengldquoAntiapoptotic and antioxidant effects of rosmarinic acid inastrocytesrdquo Pharmazie vol 60 no 1 pp 62ndash65 2005

[36] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[37] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

[14] M Foldvari ldquoNon-invasive administration of drugs throughthe skin challenges in delivery system designrdquo PharmaceuticalScience amp Technology Today vol 3 no 12 pp 417ndash425 2000

[15] AVerma S Singh R Kaur A Kumar andUK Jain ldquoFormula-tion optimization and evaluation of clobetasol propionate gelrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 666ndash674 2013

[16] M G Dodov K Goracinova M Simonoska S Trajkovic-Jolevska J T Ribarska and M D Mitevska ldquoFormulation andevaluation of diazepamhydrogel for rectal administrationrdquoActaPharmaceutica vol 55 no 3 pp 251ndash261 2005

[17] FDAGuidance for Industry Nonsterile Semisolid Dosage FormsScale-Up and Postapproval Changes Chemistry Manufacturingand Controls In Vitro Release Testing and In Vivo BioequivalenceDocumentation FDA 1997

[18] G L Flynn V P Shah S N Tenjarla et al ldquoAssessment of valueand applications of in vitro testing of topical dermatologicaldrug productsrdquoPharmaceutical Research vol 16 no 9 pp 1325ndash1330 1999

[19] A Stelmakiene K Ramanauskiene and V Briedis ldquoRelease ofrosmarinic acid from semisolid formulations and its penetra-tion through human skin ex vivordquo Acta Pharmaceutica vol 65no 2 pp 199ndash205 2015

[20] M Zilius K Ramanauskiene and V Briedis ldquoRelease ofpropolis phenolic acids from semisolid formulations and theirpenetration into the human skin in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID958717 7 pages 2013

[21] F J Ahmad M A Alam Z I Khan R K Khar and MAli ldquoDevelopment and in vitro evaluation of an acid bufferingbioadhesive vaginal gel for mixed vaginal infectionsrdquo ActaPharmaceutica vol 58 no 4 pp 407ndash419 2008

[22] L Liu H Xie X Chen et al ldquoDifferential response of normalhuman epidermal keratinocytes and HaCaT cells to hydrogenperoxide-induced oxidative stressrdquo Clinical amp ExperimentalDermatology vol 37 no 7 pp 772ndash780 2012

[23] V Armoskaite K Ramanauskiene and V Briedis ldquoEvaluationof base for optimal drug delivery for iontophoretic therapyinvestigation of quality and stabilityrdquo African Journal of Phar-macy and Pharmacology vol 6 pp 1685ndash1695 2012

[24] M-H Schmid-Wendtner and H C Korting ldquoThe pH of theskin surface and its impact on the barrier functionrdquo SkinPharmacology and Physiology vol 19 no 6 pp 296ndash302 2006

[25] K Rizi R J Green M X Donaldson and A C WilliamsldquoUsing pH abnormalities in diseased skin to trigger and targettopical therapyrdquo Pharmaceutical Research vol 28 no 10 pp2589ndash2598 2011

[26] K Cal ldquoAcross skin barrier known methods new perfor-mancesrdquo Frontiers in Drug Design amp Discovery vol 4 no 1 pp162ndash188 2009

[27] D R Drake K A Brogden D V Dawson and P W WertzldquoThematic review series skin lipids Antimicrobial lipids at theskin surfacerdquo The Journal of Lipid Research vol 49 no 1 pp4ndash11 2008

[28] G P Moss J C Dearden H Patel and M T D CroninldquoQuantitative structure-permeability relationships (QSPRs) forpercutaneous absorptionrdquo Toxicology in Vitro vol 16 no 3 pp299ndash317 2002

[29] D Touboul F Halgand A Brunelle et al ldquoTissue molecularion imaging by gold cluster ion bombardmentrdquo AnalyticalChemistry vol 76 no 6 pp 1550ndash1559 2004

[30] N Sivaranjani S V Rao andG Rajeev ldquoRole of reactive oxygenspecies and antioxidants in atopic dermatitisrdquo Journal of Clinicaland Diagnostic Research vol 7 no 12 pp 2683ndash2685 2013

[31] O Fadel K El Kirat and S Morandat ldquoThe natural antioxidantrosmarinic acid spontaneously penetratesmembranes to inhibitlipid peroxidation in siturdquo Biochimica et Biophysica ActamdashBiomembranes vol 1808 no 12 pp 2973ndash2980 2011

[32] E-R Lee Y-J Kang J-H KimH T Lee and S-G Cho ldquoMod-ulation of apoptosis in HaCaT keratinocytes via differentialregulation of ERK signaling pathway by flavonoidsrdquoThe Journalof Biological Chemistry vol 280 no 36 pp 31498ndash31507 2005

[33] M A Soobrattee V S Neergheen A Luximon-Ramma OI Aruoma and T Bahorun ldquoPhenolics as potential antiox-idant therapeutic agents Mechanism and actionsrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

[34] NAKelseyHMWilkins andDA Linseman ldquoNutraceuticalantioxidants as novel neuroprotective agentsrdquoMolecules vol 15no 11 pp 7792ndash7814 2010

[35] L P Gao H L Wei H S Zhao S Y Xiao and R-L ZhengldquoAntiapoptotic and antioxidant effects of rosmarinic acid inastrocytesrdquo Pharmazie vol 60 no 1 pp 62ndash65 2005

[36] I Chkhikvishvili T Sanikidze N Gogia et al ldquoRosmarinicacid-rich extracts of summer savory (Satureja hortensis L) pro-tect jurkat T cells against oxidative stressrdquo Oxidative Medicineand Cellular Longevity vol 2013 Article ID 456253 9 pages2013

[37] J L Martindale and N J Holbrook ldquoCellular response tooxidative stress signaling for suicide and survivalrdquo Journal ofCellular Physiology vol 192 no 1 pp 1ndash15 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom