Drug Delivery, 14:427–432, 2007Copyright c© Informa Healthcare USA, Inc.ISSN: 1071-7544 print / 1521-0464 onlineDOI: 10.1080/10717540701202960

Effect of Vehicles on Topical Application of Aloe Veraand Arnica Montana Components

Valentina Bergamante, Gian Carlo Ceschel, and Sergio MarazzitaDipartimento di Scienze Farmaceutiche, Universita di Bologna, Bologna, Italy

Celestino RonchiMonteResearch S.r.l., Bollate (Milano), Italy

Adamo FiniDipartimento di Scienza dei Metalli, Elettrochimica e Tecniche Chimiche, Universita di Bologna,Bologna, Italy

In this study two types of gels and microemulsions are investi-gated for their ability to dissolve, release, and induce the perme-ation of helenalin, a flavonoid responsible for the anti-inflammatoryactivity of arnica montana extract, and aloin, an anthrone-C-glucosyls with antibacterial activity present in aloe vera extract.The release of these agents from each vehicle was followed byHPLC, and transcutaneous permeation was examined using a mod-ified Franz cell and a porcine skin membrane. The study showedthat a microemulsion can be a good vehicle to increase the per-meation of helenalin, while the gel formulation, containing Sepigel305, proved able to reduce the release and permeation of aloin, witha consequent activity limited to the surface of application, withoutany permeation. This is in accordance with the necessity to avoidthis process, since human skin fibroblasts can metabolize absorbedaloin into a structurally related compound that increases the sen-sitivity of skin to ultraviolet light.

Keywords Aloin, Gel, Helenalin, Microemulsion, Permeation,Release

Alternative medicine, particularly herbal therapies, which in-clude a wide range of practices and therapies outside conven-tional medicine, is widespread and increasing, despite a frequentlack of scientific data in the form of controlled trials for eitherefficacy or safety. Contrary to popular belief, natural therapiesare not necessarily safe, even if topical application is considered(1). In fact when herbal extracts are topically applied, a varietyof parameters must be considered, such as effective release andpossible absorption of the active agents, driven by the vehicle:

Received 6 October 2006; accepted 24 November 2006.Address correspondence to Prof. Adamo Fini, Dipartimento di

Scienza dei Metalli, Elettrochimica e Tecniche Chimiche, Via SanDonato 15, 40127 Bologna, Italy. E-mail: adamo.fini@unibo.it

modern pharmaceutical technology is able to give convincinganswers to these questions (2).

Arnica is fairly well known in popular medicine, being avail-able as a flower essence, homeopathic remedy, and in herbalform. Arnica has been used since the seventeenth century tosoothe muscle aches and reduce inflammation, caused by a blow,a fall, or a contusion; while it is primarily for external use, it isalso known as a homeopathic remedy for muscle strain and sore-ness (3). Active components in arnica are sesquiterpene lactonesthat are known to reduce inflammation and decrease pain, thy-mol (an essential oil), flavonoids, inulin, carotenoids, and tan-nins. Anti-inflammatory effects also are attributed to helenalin,whose actions include a marked antiedemic effect that has beenconfirmed in experimental models (4). Arnica is recommendedfor external use in the form of an oil or tincture mainly by dis-persing trapped, disorganized fluids from bumped and bruisedtissue, joints, and muscles (5).

Aloe, a popular houseplant, has a long history as a multi-purpose folk remedy. Aloe gel has been used for a long time fortopical treatment of skin irritations; but aloe also can be used as abeverage and aloe products for internal use have been promotedfor constipation. Aloe extract can be taken orally as a dietarysupplement, even though it does not have FDA approval for useas a drug (6).

MATERIALS AND METHODSAloe vera dry extract (aloin content: approximately

0.2%), arnica montana glycolic extract (helenalin content:approximately 0.03%), carboxypolymethylene (Carbopol974P

©R ), polyacrylamide C13-C14/isoparaffin, and laureth 7(Sepigel 350

©R ) were supplied by Polichimica (Bologna, Italy).Diethylene glycol monoethyl ether (Transcutol P

©R ), caprylo-caproyl macrogol glycerides (Labrasol

©R ), oleoyl macrogol glyc-erides (Labrafil

©R M1944 CS), and propylene glycol monolaurate

427

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428 V. BERGAMANTE ET AL.

(Lauroglycol 90©R ) were supplied by Gattefosse (Milan,

Italy).

Gel PreparationTwo gels, containing 1% aloe vera dry extract and 4% arnica

montana glycolic extract, respectively, were developed using 1%of Carbopol 974P as a gelling agent: they are referred to as gel ACand HC, respectively. Another two gels of similar compositionin active agents (gel AS and gel HS, respectively) were devel-oped using 1% of Sepigel 305, as a gelling agent. Accuratelyweighed amounts of each gelling compound were dispersed inappropriate amounts of water, where the active agent had beenpreviously dispersed, with continuous stirring, using a porce-lain mortar and pestle until uniform consistency was achieved.Sodium hydroxide was added dropwise up to pH 7.4 (∼3 drops)in the case of Carbomer 940 P.

Microemulsion PreparationTwo aqueous microemulsions, containing 4% arnica

montana, were obtained by addition of homogenous liquid mix-tures of oil, surfactant, and cosurfactant to water phase at roomtemperature (7). A surfactant (Labrasol

©R ) was gently mixedwith a cosurfactant, respectively, Capryol 90 for microemulsion1 (M1) and Lauroglycol 90 for microemulsion 2 (M2). Aliquotsof each surfactant-cosurfactant mixture were then mixed withthe oil phase. Distilled water was added to the mixture drop bydrop under gentle agitation until a clear, isotropic and thermody-namically stable dispersion with low viscosity was obtained.Thecompositions of the microemulsions are given in Table 1 andthroughout this article are referred to as HM1 and HM2.

TABLE 1Weight percent composition of arnica montana microemulsions

HM1 HM2Components (% w/w) (% w/w)

Arnica montana glycolic extract(helenalin content 0.03%)

4.00 4.00

Labrasol©R (caprylocaproyl macrogol

glycerides)57.00 42.30

Capryol 90 (propylene glycolmonocaprylate)

9.50 —

Lauroglycol 90 (propylene glycolmonolaurate)

— 7.05

Transcutol P (diethylene glycolmonoethyl ether)

9.00 —

Labrafil©R M 1944 CS 4.50 12.30

Demineralized water (up to) 100 100

Solubility DeterminationThe aloin and helanalin equilibrium solubility was mea-

sured at 25◦C in the different vehicles. Gels and microemul-sions, prepared as described, were centrifuged at 4000 rpm. Thesurnatant, recovered and filtered (0.45 µm nylon filter, MSF,Dublin, Ireland), was essayed for the content in aloin and hele-nalin by the HPLC method described here.

Aloin and Helenalin AnalysisAloin and helenalin were determined using a reversed-

phase high performance liquid chromatography (HPLC) assay.A Dionex P580 system equipped with a variable-wavelengthUV detector UVD170S was used for this purpose. Acetonitrile(25%) and water (75%) were pumped at a flow rate of 1 mL/minthrough a Nova-Pak C18 (150 × 3.9 mm, 4 mm, Waters) at roomtemperature. The injected volume was 20 µl and the detectionwavelength was 355 nm. In these conditions, the retention timeof aloin was 6.2 min and that of helenalin 25 min. The Euro-pean Pharmacopeia (4th ed., 2002) reports an HPLC methodfor the quantification of lactone sequiterpenes in the herbal drug(arnica flower), expressed as helenalin tiglinate and calculatedusing santonin as the reference compound (8).

Release StudiesAloin and helenalin release studies for the developed gels

were performed in a diffusion cell, (9) consisting of a donorand a receptor compartment separated by a dialysis membrane(cellulose acetate, cut-off 12–14000 D). Each single formulation(2 ml) was placed in the donor compartment with a diameter of9 mm (0.636 cm2 diffusion area). The receptor compartmenthad a volume of 60 ml and was filled with 40 ml of purifiedwater maintained at 37◦C by means of a thermostated bath. Atintervals of 1, 2, 4, 8, 12, and 24 h all the volume containedin the receptor compartment was withdrawn, replaced with anequal volume of fresh solution, and assayed for the drug contentby HPLC technique.

The release kinetics can be assessed by fitting the experimen-tal data in the semiempirical equation (10):

Mt/Minf = ktn

where Mt/Minf is the fractional amount of the drug at the time t;k is a kinetic constant of the system, indicative of the rate of re-lease; and n is the release exponent indicative of the mechanismof release. Values for n and k for each system were obtained byplotting the logarithm of the fractional release against the loga-rithm of time. n and k values can be obtained graphically as theslope of the linear portion of the plot, while log k is representedby the intercept (Table 2). The values of n and k were calculatedby regression analysis.

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TOPICAL USE OF ALOE VERA AND ARNICA MONTANA 429

TABLE 2Parameters of release and permeation for aloin (A) and helanalin (H) from the vehicles.

n k J Kp (cm/h)Solubility (diffusional (cm∗h−1) (mg/cm2h−1) (permeation

Vehicle (µg/ml) exponent) (kinetic constant) (flux) coefficient)

AC7.02

sd 2.32 ∗ 10−11.26

sd 3.25 ∗ 10−21.21 × 10−5

sd 1.14 ∗ 10−71.72

sd 4.12 ∗ 10−24.05 ∗ 10−4

sd 2.36 ∗ 10−6

AS5.57

sd 1.12 ∗ 10−10.82

sd 2.21 ∗ 10−21.56 × 10−4

sd 2.32 ∗ 10−63.28 ∗ 10−3

sd 4.56 ∗ 10−56.56 ∗ 10−7

sd 4.99 ∗ 10−9

HC0.70

sd 3.12 ∗ 10−20.86

sd 2.65 ∗ 10−22.59 ∗ 10−4

sd 3.26 ∗ 10−68.44 ∗ 10−3

sd 3.63 ∗ 10−51.21 ∗ 10−5

sd 6.39 ∗ 10−7

HS0.69

sd 4.23 ∗ 10−20.66

sd 3.29 ∗ 10−22.88 ∗ 10−4

sd 7.69 ∗ 10−79.27 ∗ 10−2

sd 9.63 ∗ 10−57.72 ∗ 10−5

sd 3.26 ∗ 10−7

HM17.80

sd 6.14 ∗ 10−10.71

sd 3.11 ∗ 10−29.12 ∗ 10−4

sd 3.69 ∗ 10−66.77

sd 1.25 ∗ 10−15.64 ∗ 10−3

sd 9.84 ∗ 10−6

HM27.59

sd 7.12 ∗ 10−10.92

sd 2.36 ∗ 10−26.11 ∗ 10−4

sd 9.63 ∗ 10−710.03

sd 2.26 ∗ 10−18.36 ∗ 10−3

sd 3.36 ∗ 10−5

Tissue PreparationThe porcine skin is used mainly for in vitro experiments be-

cause it is similar to the human epidermis, as demonstratedby the fact that the Differential Scanning Calorimetry (DSC)of the porcine and human horny layer are very similar andpresent the same peaks and porcine skin offers resistance topermeation similar to human skin (11–13). Full-thickness skinwith a fair amount of underlying connective tissue was sur-gically removed from the ears of a freshly killed male pig(30–50 Kg) obtained from a local slaughter house (CLAI,Imola, Italy) under the supervision of a veterinary doctor. Theskin was placed in pH 7.4 ice-cold phosphate buffered saline(PBS). The connective tissue of the skin was carefully re-moved using fine-point forceps and surgical scissors until a1-mm thick membrane was obtained. The cleaned membranewas then placed in ice-cold PBS until it was set in the diffusioncells (14). The thickness of the porcine skin used in the experi-ment was measured by means of an electronic calliper; the skinwas 1.0 ± 0.1 mm.

In Vitro Permeation StudyAll the in vitro permeation studies were carried out in Franz’s

diffusion cells (diameter: 9 mm; diffusion area: 0.64 cm2 ) (15,16). The receptor compartment had a volume of 4.8 ml andwas filled with a water:ethanol 6:4 (v/v) solution maintainedat 37◦C by means of thermostated water circulating in a jacketsurrounding the cells. The solution in the receptor compartmentwas continuously stirred at 600 rpm using a Teflon coatedmagnetic stirrer. Drug permeation tests were carried out using2 ml of each formulation, placed in the donor compartment.Porcine skin was used as membrane between the donor andreceptor compartment of the cells (17, 18). Samples from the

donor receptor (2 ml) were withdrawn at regular intervals,filtered, and assayed by HPLC technique. The solution in thereceptor compartment was restored after each withdrawal withan equal volume of fresh solution.

Permeation through the skin membrane can be considered asa passive diffusion process and can be described by Fick’s lawequation:

Js = dQr/Adt

where J is the steady-state flux in mg/cm2 per h; dQr is thechange in quantity of material passing through the membraneinto the receptor compartment and expressed in milligrams, Ais the active diffusion area in cm2; and dt is the change in timein hours.

RESULTS AND DISCUSSIONHerbal drugs are an important alternative to chemicals in the

therapy of minor disorders and are widely employed in topicalformulations, where the presence of natural agents often rep-resents an additional value for marketing purposes. However,the interest in aloe and arnica extract is based on the relevantefficacy for the active agents aloin and helenalin for their anti-inflammatory activity. But since aloin has been demonstrated tobe photocytotoxic if absorbed (19,20), topical application of thiscompound must be carried out so that the therapeutic action isperformed only on the skin surface.

Arnica montana and Aloe vera extracts contain a large num-ber of components to treat symptoms of human diseases or toimprove specific aspects of the body condition. The main activecompounds present in their extracts are, respectively, helenalin(H), a flavonoid responsible for the anti-inflammatory activityof arnica montana, and aloin (A), an anthrone-C-glucosyls with

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430 V. BERGAMANTE ET AL.

FIG. 1. Release profiles of helenalin and aloin from tested formulations.

antibacterial activity present in aloe vera. They were formulatedas two gels and two microemulsions.

The composition of the two gels is simple: they contain thesame low (1% w/w) amount of two different gelling compoundsand only one aqueous phase. Carbopol is a polycarboxylic acidneeding a small amount of sodium hydroxide to start a partialionization that in turn promotes the gel formation (21). Sepigel isa more complex and nonionic gel promoter and does need NaOH.Sepigel 305, also known as Farcosgel polyacrylamide, and C13-14/isoparaffin (and) laureth-7 appears as a fluid, opalescent, yel-lowish dispersion in a 2% aqueous solution: it is classified asa gelling and thickening agent, excellent in the preparation ofaqueous gels (22).

Microemulsions are quaternary systems consisting of an oilphase, a water phase, a surfactant, and a co-surfactant. Thesesystems spontaneously form when the components are mixedtogether and possess specific physicochemical properties suchas transparency, optical isotropy, low viscosity, and thermody-namic stability. Microemulsion formulations also display im-proved transdermal delivery properties, in vitro and in vivo (23).In addition to an aqueous phase, microemulsions contain thesame oily phase (Labrafil) and the same surfactant (Labrasol),though at different percentages (Table 1).

Labrasol (HLB 14), a biocompatible and biodegradable PEGderivative, is a nonionic emulsifier, easily soluble in water, andused in oral formulations for its high tolerance and low toxicity.Transcutol displays a strong absorption enhancing effect, usedas a cosurfactant in microemulsions (24). M1 and M2 differ intheir cosurfactants (Capryol 90 for M1 and Lauroglycol 90 forM2), even though of very close molecular structure (caprylate C8vs. laurate C12; diethylene vs propylene). The most remarkabledifference is the presence of transcutol in M1, which behaves asa penetration enhancer.

This formulation has received a great deal of attention in re-cent years for its advantages in transdermal delivery of drugs:a large amount of drug can in fact be incorporated in the for-mulation because of its high solubilizing capacity, especially ofhydrophobic drugs, due to the presence of a hydrophobic phase.A second advantage is that the permeation rate of the drug frommicroemulsion may be increased: modifying the internal phase,changing its composition, and thus adjusting its property.

Aloin and helenalin are well-defined chemical components,with different hydrophillic contributions (see Figure 1). Inaccordance with these structures, aloin was found to be moresoluble than helenalin in the two gels. Whereas helenanlinappears not to be affected by the composition of the gel,important differences were found for aloin, which in gel ASdisplayed a notably higher solubility. Aloin (25) proved tobe more soluble in a poorly polar solvent: solubility almostdoubles, passing from aqueous ethanol or propylene glycol(1:1) to mixtures where the organic solvent is 90%.

The presence of the drug in these systems as a solute providesthe necessary concentration gradient for absorption: however,though higher in microemulsions, aloin does not appear as highas expected, despite the presence of an oil phase in this formula-tion. It cannot be excluded that in microemulsion HM2, due tothe high concentration of Labrasol, the drug can be distributedover three different phases: the dispersed phase, the continuousphase and the surfactants micelles.

Release from FormulationsEach formulation was tested as far as release is concerned

through a dialysis membrane that should not offer resistanceto the crossing of the drug. Figure 1 shows the results of re-lease for the different formulations as a function of time. Theprofile linearly increases during the period examined for all

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TOPICAL USE OF ALOE VERA AND ARNICA MONTANA 431

the formulations. After 8 h it is comparable for the differentmicroemulsions containing helenalin, reaching 5000 µg, whereas the total release is ∼5- fold lower for the two gels HC and HS.On the contrary, aloin was not released at all from its gels. GelsAC and AS hinder diffusion of aloin, probably through hydrogenbonds between the molecule and the aqueous environment; dif-fusional difficulties, due to semisolid consistency of the gelifiedsystems, appear only to occur in the case of aloin.

The comparison between solubility in the medium and themedium’s ability to release the drug suggests that the main driv-ing force for release is the concentration gradient of the dissolveddrug between the formulation and membrane. As a consequence,a rapid and consistent release from a topical formulation is bestobtained by high solubility than rapid dissolution rate. However,all formulations are saturated systems and release concerns onlythe drug originally dissolved inside the formulation. Finally allthe formulations allowed the release of helenalin with a rate andextent according to their nature, but without differences betweensimilar physical forms. This fact could not represent an obstacleto the supply of helenalin, when present in a topical formulationfor transdermal absorption. Release of aloin is inhibited as theaffinity to the vehicle becomes greater due to a slow release ofthe drug and/or a poor transfer outside the vehicle.

Aloin and Helenalin PermeationThe overall process of trandermal absorption of a drug is

largely affected by the physicochemical characteristics of thepenetrant and of the vehicle in which the drug is carried. Ad-ditives of topical formulations modulate the diffusion and therelease of the active principle through the matrix and markedlyaffect the penetrant properties and absorption of the active agent,and thereby affect drug potency or effectiveness.

Figure 2 shows that microemulsions (M1 > M2) induce bet-ter permeation than the two gels (HC > HS), probably due tothe presence of an oily phase, which allows a more intimate

contact with the absorption membrane. Permeation ranges from∼30 for HS to ∼110 µg after 8 h application. The permeationprofile is not linear but reaches fair linearity after 2 h. Moreover,the surfactant/cosurfactant system in the microemulsions mayreduce the diffusional barrier of the stratum corneum by actingas permeation enhancers and wettability agents.

Gels containing aloin do not allow either release or absorp-tion (see Figure 3). We previously pointed out that in the caseof aloin it is desirable to minimize its absorption to restrict thedrug to the diseased surface of application, avoiding potentialside-effects associated with absorption. This goal appears tobe achieved using the gels of the proposed formulations. Aloinskin permeation was inhibited since its high affinity with thevehicle produces not only a slow release, but also a poor transferfrom the vehicle to the skin.

Fitting experimental data by means of the following equation

Mt/Minf = ktn

kinetic constant for the release could be evaluated differentlyby almost one order of magnitude passing from HM1 and HM2to gel HS and gel HC to Gel AC; the diffusional exponent ishigher than 0.5, suggesting that a combination of mechanismsoperates for the release of the drug from the different vehicles.Flux values have a similar order of magnitude for the systemsAC, HC, and HC, whereas they are the lowest for AS. The flux ofhelenalin from the two microemulsions is decisively higher. Thecorresponding Kp value for this last system is thus the lowestone among all the systems studied, indicating that it could bethe preferred formulation in the case of only activity on the skinsurface for aloin, topically applied.

On the contrary, both microemulsions (HM1 and HM2)proved useful when helenalin permeation is needed, consider-ing that even larger amounts of the drug can be incorporated inthe formulation due to the high solubilizing capacity and that

FIG. 2. Permeation profiles of helenalin and aloin from tested formulations.

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432 V. BERGAMANTE ET AL.

FIG. 3. Permeation profiles of Helenalin and Aloin from gels.

the permeation rate from the microemulsion may be increased.The affinity of a drug to the internal phase in microemulsionscan be easily modified using different components, changingtheir amount in the microemulsion, or adjusting its property.Microemulsions therefore appear to be an ideal vehicle in favor-ing the partitioning of a dissolved active agent into the stratumcorneum, when absorption is desirable.

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