Interaction of β-caryophyllene and β-caryophyllene oxide with phospholipid bilayers: Differential scanning calorimetry study

Accepted Manuscript Title: Interaction of ␤-Caryophyllene and ␤-Caryophyllene oxide with phospholipid bilayers: Differential scanning calorimetry study Author: Maria Grazia Sarpietro Antonella Di Sotto Maria Lorena Accolla Francesco Castelli PII: DOI: Reference:

S0040-6031(14)00545-0 http://dx.doi.org/doi:10.1016/j.tca.2014.11.029 TCA 77086

To appear in:

Thermochimica Acta

Received date: Revised date: Accepted date:

30-7-2014 27-11-2014 28-11-2014

Please cite this article as: Maria Grazia Sarpietro, Antonella Di Sotto, Maria Lorena Accolla, Francesco Castelli, Interaction of rmbeta-Caryophyllene and rmbetaCaryophyllene oxide with phospholipid bilayers: Differential scanning calorimetry study, Thermochimica Acta http://dx.doi.org/10.1016/j.tca.2014.11.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Interaction of β-Caryophyllene and β-Caryophyllene oxide with phospholipid bilayers: differential scanning calorimetry study Maria Grazia Sarpietroa*[email protected], Antonella Di Sottob, Maria Lorena Accollac, Francesco Castellia

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Dipartimento di Scienze del Farmaco, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy

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Dipartimento di Fisiologia e Farmacologia, Sapienza – Università di Roma, P.le Aldo Moro 5, 00185 Rome, Italy c

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Dipar mento di Scienze della Salute, Università “Magna Græcia” di Catanzaro, Campus Universitario “S. Venuta”, Viale S. Venuta, 88100 Germaneto (CZ), Italy

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corresponding author. Tel.: +39 095 7385099; fax: +39 095 580138.

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Highlights

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The interaction of β-caryophyllene and β-caryophyllene oxide with model membrane was studied;

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The medium influence on membrane absorption was also assessed;

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The differential scanning calorimetry technique was used;

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The interaction is controlled by compound structural characteristics;

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Abstract

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The membrane absorption is allowed by the lipophilic medium.

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The ability of the flavouring sesquiterpenes β-caryophyllene and β-caryophyllene oxide to be absorbed into the cell has been investigated by an in vitro biomembrane model of multilamellar vesicles. Dimyristoylphosphatidylcholine was used as a constituent of the biomembrane and permitted the experiments to be carried out at 37.0 °C, close to body temperature. Differential scanning calorimetry was applied for measuring the effect of the compounds on the thermotropic behaviour of the vesicles. Studies were also carried out in order to characterize the interference of an aqueous medium or a lipid carrier with the uptake of the substances by the biomembrane. The results showed that both sesquiterpenes influenced the behaviour of the multilamellar vesicles, with β-caryophyllene producing a larger effect than β-caryophyllene oxide. The aqueous medium hindered the absorption of the test substances, probably because of their low water solubility; on the contrary, the lipophilic medium strongly favoured their uptake. Present results provide preliminary

information on the bioavailability of β-caryophyllene and β-caryophyllene oxide and allow us to expect their uptake through cell membranes. Moreover, these results could be of importance for future design of lipophilic delivery systems for these sesquiterpenes.

Keywords: β-Caryophyllene, β-caryophyllene oxide, differential scanning calorimetry, MLV

1. INTRODUCTION β-Caryophyllene is a natural biciclic sesquiterpene (Figure 1), widely occurring in the essential oils

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from different spice and medicinal plants, such as oregano (Origanum vulgare L.), cinnamon (Cinnamomum spp.), clove (Eugenia caryophyllata L.), Cannabis sativa L., Zingiber nimmonii (J.

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Graham) and black pepper (Piper nigrum L.) [1,2]. Likewise, β-caryophyllene oxide (Figure 1), an

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epoxide derivative from β-caryophyllene, is a component of many essential oils, especially of clove,

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citrus (Citrus spp.), basil (Ocimum basilicum L.) and hop (Humulus lupulus L.) [3-6]. Because of

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their woody and spicy odour, both β-caryophyllene and β-caryophyllene oxide are used as flavours

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and fragrances since the 1930s [7, 8] and are enclosed in the European list of flavourings

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substances, carrying the identification numbers of FL no. 01.007 and FL no. 16.043, respectively

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[9].

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Recently, these sesquiterpenes have attracted attention due to their several interesting properties. βcaryophyllene has been reported to induce anxiolytic-like and local anaesthetic effects [10] and to

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prevent colitis and nephrotoxicity through a CB2 receptor-dependent pathway [11, 12]. Also it

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exerted neuroprotective effects by modulating the expression of inflammatory mediators [13]. Likewise, β-caryophyllene oxide exhibited analgesic and anti-inflammatory activities [14]. Both

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sesquiterpenes have also been studied as chemopreventing agents, resulting effective antimutagenic and antiproliferative agents [7, 15-17]. β-Caryophyllene was also shown to potentiate the anticancer effect of a-humulene, isocaryophyllene and paclitaxel against some cancer cells, due in part to the alteration of the membrane permeability [18]. Accordingly, da Silva and coworkers [19] found that

this sesquiterpene, associated to the synergism of other components of the essential oil from Zanthoxylum rhoifolium Lam leaves, exhibited antitumor and immunomodulatory activity in vivo. The major limit for the application of these sesquiterpenes in pharmaceutical field is their poor water-solubility [16, 20] which may substantially decrease their bioavailability and efficacy. The pharmacokinetic behaviour of a compound strictly depends on its binding affinity to phospholipid bilayers and on its ability to be uptaken. On the basis of these findings, in present paper the interaction of the sesquiterpenes β-caryophyllene

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and β-caryophyllene oxide with the phospholipid bilayers has been evaluated by using an in vitro

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biomembrane model of dimyristoyl phosphatidylcholine multilamellar vesicles (DMPC MLV). The non perturbative technique of differential scanning calorimetry (DSC) was applied for measuring

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the effect of the molecules on the thermotropic behaviour of DMPC MLV [21, 22]. Experiments

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were also carried out in order to characterize the interference of the aqueous or lipid medium with

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the uptake of the test substances through the biomembrane model [23].

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2.1. Materials

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2. MATERIAL AND METHODS

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1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) (purity = 99%) was supplied by

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Genzyme Pharmaceuticals (Liestal, Switzerland). Methanol and chloroform were of LC grade and

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were bought from VWR (Darmstadt, Germany). The test compounds β-caryophyllene (purity ≥ 98.5%) and β-caryophyllene oxide (purity 99%) were purchased from Sigma-Aldrich Co (St. Louis,

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MO, USA).

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Calorimetric analyses were performed using a Mettler Toledo STARe system (Greifensee, Switzerland) equipped with a DSC-822e calorimetric cell, and employing Mettler TA-STARe software (8.1 version). The sensitivity was chosen automatically as the maximum possible by the calorimetric system, and the reference pan was filled with the 50 mM Tris buffer (pH 7.4). The calorimetric system was calibrated, in terms of temperature and enthalpy changes, following the

procedure of the DSC 822 Mettler TA STARe instrument, using indium and palmitic acid (purity ≥ 99.95% and ≥ 99.5%, respectively; Fluka, Buchs, Switzerland). 2.2. MLV preparation MLV were prepared in absence and in presence of β-caryophyllene and β-caryophyllene oxide at different molar fractions (0.015; 0.030; 0.045; 0.060; 0.090; 0.120) in accord to a published method [24]. Briefly, stock solutions of DMPC, β-caryophyllene and β-caryophyllene oxide dissolved in chloroform/methanol (1:1, v/v) were prepared. Aliquots of the DMPC solution were placed in glass

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tubes where aliquots of the test compound solutions were added, in order to have the chosen molar

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fractions. The solvents were removed under a nitrogen stream at 37.0 °C, to obtain a thin lipid film containing β-caryophyllene or β-caryophyllene oxide. Afterwards, the samples were freeze dried

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(FreeZone® 200 2.5 Liter Freeze Dry Systems, Labconco) to remove eventual solvent residues. 168

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μL of 50 mM Tris(hydroxymethyl)-aminomethane (Tris) (pH = 7.4) was added to the film, then the

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samples were heated at 37.0 °C for 1 min and vortexed for 1 min, for three times and, finally, kept

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2.3. MLV/compounds interaction

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at 37.0 °C for 1 h.

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A volume of 120 μL of MLV, alone or containing the test compound, was put into the calorimetric

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pan, which was hermetically sealed and submitted to the DSC analysis. Three cooling–heating

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cycles were performed from 5.0 to 37.0 °C, at 2.0 °C/min and 4.0 °C, respectively. Each experiment was carried out in triplicate. All the samples, after the calorimetric scans, were extracted from the

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pan and aliquots were used to determine the exact amount of phospholipids by the phosphorous

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assay [25]. The third heating scan was used for the calculation of the thermotropic parameters of the phase transitions using the Mettler STARe software. 2.4. Uptake experiments This experiment was run to evaluate the uptake of β-caryophyllene oxide and β-caryophyllene by the biomembrane model in a hydrophilic medium [23]. To perform the test, 120 μL of MLV were put in contact with a fixed amount of the test substance (weighted as a powder in the bottom of the

DSC pan) in order to obtain a 0.09 molar fraction of compound with respect to the phospholipids. The weighing of the sample and the contact with the MLV were made at 22.0 °C. Immediately after, the aluminum pan was hermetically closed and the sample was kept at 5.0 °C for two minutes and submitted to the following calorimetric steps: (1) a scan performed from 5.0 to 37.0 °C, at the rate of 2.0 °C/min, to detect the eventual interaction of the test substance with the MLV, due to its dissolution and to its successive migration through the aqueous medium until the eventual absorption through the phospholipid bilayers of MLV; (2) the stay of test compounds for 1 h in

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isothermal mode (37.0 °C), when the liposomal system is in a disordered state, in order to permit

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that the substance, dissolved in the medium, further migrates toward the MLV surface, is uptaken by them and interacts with the phospholipid bilayers; (3) a cooling scan from 37.0 °C to 5.0 °C, at

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the rate of 4.0 °C/min, to bring the sample back to the starting temperature of the successive cycle

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as soon as possible but allowing the thermodynamic equilibrium to be reached (the rate was chosen

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after several tests).This procedure was continuously run at least ten times. Each experiment was

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carried out in triplicate. The analysis was performed also on MLV without compound. After the

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calorimetric scans, aliquots of the samples were used to determine the exact amount of

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phospholipids by the phosphorous assay [25].

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2.5. Transmembrane transfer experiments

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Transmembrane transfer experiment was carried out to verify if a lipophilic medium (lipid MLV), where the test compound was molecularly dispersed, could improve its uptake by the biomembrane

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model. To perform the experiment 60 μL of MLV prepared in the presence of β-caryophyllene or β-

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caryophyllene oxide at 0.09 molar fraction (loaded MLV) and 60 μL of an equimolar dispersion of MLV made of pure DMPC (unloaded MLV) were put in contact in a 160 μL DSC pan, at 22.0 °C, hermetically sealed kept at 5.0 °C for two minutes, and submitted to the DSC analysis following the same procedure reported in the previous section. The experiment was carried out in triplicate. 3. RESULTS AND DISCUSSION

Differential scanning calorimetry was used to evaluate the effect of the sesquiterpenes βcaryophyllene and β-caryophyllene oxide on the thermotropic behaviour of DMPC MLV. With the increase of temperature, different states take place in DMPC MLV: the tilted gel phase (Lβ), the ripple phase (Pβ) and the liquid-crystalline phase (Lα). The passage from the gel phase to the ripple phase occurs at a specific temperature (16.1 °C) called pretransition temperature, evidenced by a socalled pretransition peak, and the passage from the ripple phase to the liquid-crystalline phase occurs at the so called main transition temperature (25.0 °C, Tm), evidenced by a main transition

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then variations in the pretransition and main transition peaks occur [27].

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peak [26]. If a molecule inserts in the phospholipid bilayers of the MLV and interact with them,

The comparison between the calorimetric curve of MLV in the absence and presence of increasing

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molar fractions of β-caryophyllene or β-caryophyllene oxide demonstrated the ability of the

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compounds to interact with the MLV phospholipid bilayers (Figure 2A and 2B). In particular, in the

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presence of β-caryophyllene, the MLV calorimetric curves are characterized by the absence of the

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pretransition peak and by a complex double-peak with one sharp and one broad component. The

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sharp component shifts to lower temperatures while its height is reduced, as the concentration of β-

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caryophyllene increases. This behaviour could be caused by a non-uniform compound distribution

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in the bilayers. The presence of the broad component could be due to a seclusion of a group of

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phospholipid molecules that are rich in β-caryophyllene. These secluded phospholipid molecules are prevented from participation in the cooperative transition because β-caryophyllene molecules

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hindrance the unison melting of DMPC molecules. The sharp component could represent a

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cooperative melting of DMPC lipid molecules with fewer β-caryophyllne molecules present; thus each lipid is in contact with other lipids and undergoes the transition as a unison. In other words, if this is indeed the case, a phase separation in the bilayer occurs: one phase rich in β-caryophyllene and the other poor in β-caryophyllene [28]. The presence of β-caryophyllene oxide caused the disappearance of the pretransition peak, the broadening of the main transition peak together with the lowering of the phase transition temperature. The effects caused by the presence of β-caryophyllene

and β-caryophyllene oxide on the transition temperature (Figure 3A), peak width at half height (ΔT1/2) (Figure 3B) and enthalpy change (ΔH) (Figure 3C) have been plotted as a function of the molar fraction of compound, permitting to obtain important information on the interaction between the tested compounds and the MLV phospholipid bilayers. As both β-caryophyllene and βcaryophyllene oxide molar fractions increase, a decrease of the transition temperature occurs. However,the transition temperature decrease due to the presence of β-caryophyllene is greater than that caused by β-caryophyllene oxide. This behaviour demonstrates that the compounds interact

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with MLV producing a fluidising effect and following the Van’t Hoff equation [29] which predicts

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an almost linear relationship between the lowering of solvent (phospholipid) melting temperature and the concentration of the solute (test compound) in the solvent (phospholipid). β-caryophyllene

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causes a greater fluidising effect than β-caryophyllene oxide. The phase transitions in phospholipid

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are cooperative phenomena; in fact, the behaviour of a molecule in one phase depends on the state

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of the other molecules around it. The sharpness of the transition is dependent upon the number of

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the molecules that cooperate in the transition. The broadening of the phase transition, and thus the

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increase of the ΔT1/2, are a sign that a compound molecules reduces the cooperativity of the phase

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transition as they intercalate into the phospholipid bilayers affecting the number of phospholipid

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molecules that a single phospholipid molecule can influence [30, 31]. Figure 3B, shows that both,

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β-caryophyllene and β-caryophyllene oxide, cause the decrease of the phospholipid cooperativity; with a much greater effect exerted by β-caryophyllene. The enthalpy change values are shown in

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figure 3C. Both of the compounds cause the decrease of the ΔH but with different extent; in fact, β-

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caryophyllene oxide causes only a small decrease whereas β-caryophyllene causes a strong decrease. This behaviour indicate that β-caryophyllene oxide molecules intercalate among the flexible chains of the phospholipids as “interstitial impurities” without lowering the stability of the lipid membrane; differently, β-caryophyllene oxide acts as an “substitutional impurity” altering the packing of the phospholipid and the stability of the bilayers [32, 33].

β-caryophyllene exerts a greater effect than β-caryophyllene oxide on all the calorimetric parameters considered. These observations suggest a different localization of the two compounds in the phospholipid bilayers and, in particular, they indicate that β-caryophyllene oxide could be preferentially located close to the lipid head groups [34] with the oxide group oriented towards the surrounding choline groups of the phospholipids. Whereas, the strong decrease of the Tm, and ΔH and the increase of the ΔT1/2, caused by β-caryophyllene suggest that the compound is located among the hydrophobic chains.

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For the uptake experiments in a hydrophilic medium the tested compounds were separately weighed

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in the calorimetric pan and MLV were added, then the sample was submitted to calorimetric analysis. Calorimetric analysis was performed also on MLV without compound and it showed that

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calorimetric curve remained unchanged in all the scans (data not shown). The data of MLV put in

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contact with the sample are shown in Figure 4 and, for comparison, the curve of MLV without

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compound and the curve of MLV prepared with 0.09 molar fraction of compound (reference curve,

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R, representing the maximal interaction of the tested compound with lipid bilayers) are reported.

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The variations of the calorimetric curves indicate that the interaction of the test substances with the

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MLV occurs. In fact, when β-caryophyllene is present, the pretransition peak gradually decreases

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and then disappears; the main transition peak shifts towards lower temperature till approaching the

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reference curve. With regard to β-caryophyllene oxide, the pretransition peak gradually disappears whereas the main transition peak moves towards lower temperatures and in the tenth scan it splits

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into two components similarly to the reference curve. The effect has been represented graphically

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by plotting the transition temperature of DMPC MLV put in contact with β-caryophyllene or βcaryophyllene oxide, as a function of the calorimetric scan (Figure 5). The value R should be obtained if a complete uptake of compound (at 0.09 molar fraction) through the MLV bilayers occurs. It is evident that the compounds, when tested in a hydrophilic medium, caused a decrease of the transition temperature, with a stronger effect of caryophyllene oxide. This behaviour indicates that, even if not completely, they are uptaken by the biomembrane model. The calorimetric curves

of the transmembrane transfer experiments, for which unloaded and loaded MLV (with 0.09 molar fraction of test compounds) were put in contact at increasing incubation times are showed in Figure 6. For loaded MLV, the pretransition peak decreases and the transition peak shifts towards lower temperature. The following curves do not show the pretransition peak whereas the transition peak gradually shifts towards lower temperature. The behaviour is better visible when the transition temperature is plotted as a function of the calorimetric scan (Figure 7). The transition temperature decreases approaching the value R (transition temperature of the reference curve). It indicates that

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the compound transfer from loaded to unloaded MLV and then that the lipophilic carrier could

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enhance the absorption of β-caryophyllene and β-caryophyllene oxide by the biomembrane. In conclusion, β-caryophyllene and β-caryophyllene oxide are able to interact with MLV DMPC

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biomembrane model with a higher effect of the first compound. The effect caused by the two

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compounds on the calorimetric parameters indicate a different localization of the compounds in the

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phospholipid bilayers, with β-caryophyllene oxide locating next to the phospholipid head group and

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β-caryophyllene in the hydrophobic region of phospholipid. The medium influences the absorption

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of the test substances: the process is hindered by the aqueous medium, probably because of the low

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water solubility of the sesquiterpenes, particularly for β-caryophyllene oxide; conversely, the

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lipophilic medium strongly favourites their uptake. Present results provide preliminary information

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on the bioavailability of β-caryophyllene and β-caryophyllene oxide and allow us to expect their uptake through cell membranes. This is an important outcome as interaction with the membrane

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components, permeation of phospholipid bilayers and uptake into cells are essential factors so that a

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compound could produce a biological effect. Furthermore, the ability of β-caryophyllene and βcaryophyllene oxide to diffuse across DMPC MLV suggests that liposomal formulations could be a future goal to be achieved for further therapeutic application of these substances, in order to exceed the limit of poor solubility in biological fluids.

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Figure legends

Figure 1. Structure of (A) β-caryophyllene and (B) β-caryophyllene oxide.

Figure 2. Calorimetric curves, in heating mode, of MLV prepared with increasing molar fractions of (A) β-caryophyllene and (B) β-caryophyllene oxide. Figure 3. Effect of β-caryophyllene and β-caryophyllene oxide on (A) transition temperature (Tm),

PT

(B) peak width at half height (ΔT1/2 ), and (C) enthalpy change (ΔH) as a function of the compounds molar fractions.

RI

Figure 4. Calorimetric curves, in heating mode, of MLV left in contact with (A) β-caryophyllene

SC

and (B) β-caryophyllene oxide at increasing time. Curves R belong to MLV prepared with 0.09

U

molar fraction of test compounds.

N

Figure 5. Transition temperature of MLV left in contact with β-caryophyllene and β-caryophyllene

A

oxide, as a function of the calorimetric scans. Values r belong to MLV prepared with 0.09 molar

M

fraction of compounds.

D

Figure 6. Calorimetric curves, in heating mode, of unloaded MLV left in contact with (A) β-

TE

caryophyllene and (B) β-caryophyllene oxide loaded MLV at increasing time. Curves R belong to MLV prepared with 0.045 molar fraction of test compounds.

EP

Figure 7. Transition temperature of unloaded MLV left in contact with β-caryophyllene and β-

CC

caryophyllene oxide, as a function of the calorimetric scans. Values r belong to MLV prepared with

A

0.045 molar fraction of compounds.

TE

EP

CC

A D Figure 1

Fig. 2 B

PT

RI

SC

U

N

A

M

A

25

A 24

Tm (°C)

23

22

21

-cariophyllene -cariophyllene oxide

20 0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

PT

molar fraction

10

B

8

RI

-caryophyllene -caryophyllene oxide

7

SC

6 5

U

4 3 2

N

T1/2 (peak width at half height)

9

0 0.02

0.04

M

0

A

1

0.08

0.1

0.12

0.14

C

D

-15

0.06

molar fraction

-cariophyllene -cariophyllene oxide

A

CC

H (J/mmol)

EP

TE

-20

-25

-30

-35 0

0.02

0.04

0.06

0.08

molar fraction

Figure 3

0.1

0.12

0.14

TE

EP

CC

A D

PT

RI

SC

U

N

A

M

Fig. 4

Tm (°C)

25

24

23

-caryophyllene -caryophyllene oxide

22

0

2

4

6

8

10

R

PT

calorimetric scans

A

CC

EP

TE

D

M

A

N

U

SC

RI

Figure 5

Fig. 6

Tm (°C)

25

24

23

-caryophyllene -caryophyllene oxide 22 0

2

4

6

8

10

R

calorimetric scans

A

CC

EP

TE

D

M

A

N

U

SC

RI

PT

Figure 7