The Essential Oil of Thymbra capitata and its Application as A Biocide on Stone and Derived Surfaces

Many chemicals used nowadays for the preservation of cultural heritage pose a risk to both human health and the environment. Thus, it is desirable to find new and eco-friendly biocides that can replace the synthetic ones. In this regard, plant essential oils represent effective alternatives to synthetic substances for the preservation of historical monuments. Thymbra capitata (syn. Thymus capitatus) is a medicinal and aromatic plant growing in the Mediterranean area and endowed with important pharmacological properties related to its essential oil. Among them, the antimicrobial ones make the T. capitata essential oil an ideal candidate for industrial applications; for instance, as biocide for the inhibition and elimination of biological patinas of cyanobacteria and green algae on historical monuments. In the present work, we studied the chemical composition of the essential oil from T. capitata growing in Malta by gas chromatography-mass spectrometry (GC/MS). The major volatile component is the phenolic monoterpene carvacrol (73.2%), which is capable of damaging the cytoplasmic membrane and to interfere both in the growth curve and in the invasive capacity, though the contribution of minor components γ-terpinene and p-cymene cannot be disregarded. For the oil application on the stone surface, Pickering emulsions systems were prepared with an essential oil/water 1:3 mass ratio stabilized with kaolinite at 4 mass% in the presence of Laponite®; this allowed to limit the fast volatility of the oil and guaranteed a better application and an easier removal from the artefacts attacked by biodeteriogens both indoor and outdoor. This formulation caused the elimination of biodeteriogens from treated surfaces without residuals or films on artworks surface, and the effect was retained up to four months.


Introduction
The biological properties of the plant species of the genera Thymus, Thymbra, and Satureja were known from the ancient times by the Egyptians, Greeks, and Romans [1][2][3]. These civilities used the plant and their essential oils for many health problems and therapies. Plinio The Old (I century B.C) knew the properties of their essential oils and wrote different chapters in the historical book "Naturalis Historia". The above genera are part of the complex Lamiaceae family [4][5][6] and are particularly prolific of chemotypes that rise from the adaptation to various environmental conditions. purposes, so far, but using organic products such as agar-agar and funori as application matrix (gelling polysaccharides that have good application, but cost as much as essential oils) [51]. The method we present here has a simple application even in vertical surfaces, with a low cost. Moreover, the essential oil of T. capitata showed a very interesting biodeteriogenic action when compared with other essential oils of Thymus spp.
The essential oil chemical profile herein described for a wild population growing in Malta resulted consistent with that previously reported by Merino et al. [53] for a Spanish accession. Indeed, the latter was characterized by carvacrol (73.8%), p-cymene (9.2%), and γ-terpinene (5.3%).
Generally speaking, the main problems in the usage of essential oils on outdoor stone surfaces are attributable to their high volatility, low persistence, and difficult application. Consequently, we decided to obtain a physic support suitable for a correct use by preparing Pickering emulsion systems.

Pickering Emulsion
Pickering emulsions are used in the industrial fields, but, recently, their application in other sectors has drastically increased. Pickering emulsions are valid alternatives to surfactants for the formation of an emulsion. The emulsions are made up of fine particles that form a shell around the oil drop or water drop [57]. Water-in-oil (W/O) emulsions are widely used for different "soft" matter applications: pharmaceutical, food, personal care, agriculture and, as described in the present paper, in the restoration of cultural heritage [58][59][60]. Today, the use of Pickering particles as stabilizers for emulsions has received attention owing to their ability to adsorb irreversibly at the liquid/liquid interface [61,62]. Pickering stabilization happens when particles assemble at the water-oil (W/O) interface making a mechanical (steric) barrier that protects the emulsion droplets against coalescence [63]. A correct Pickering stabilization foresees that the dimensions of the adsorbed particles are at least an order of magnitude smaller than the emulsion droplet size [64,65]; smaller particles will give a higher grouping and more homogeneous layer at the interface preventing coalescence. Particles size is also directly proportional to desorption free energy as it can be calculated by the following formula: where r is the particle radius, γ ow is the interfacial tension between oil and water phases, and θ is the three phases contact angle.
In this work, for an application on the stone surfaces, several Pickering emulsions were prepared by mixing the T. capitata essential oil and water, in a ratio of 1:3, with different clays: kaolinite (Kao), sepiolite (Sep), and Laponite ® (Lap), in a wide concentration range. Nanoclays were considered because they are promising tools in cultural heritage conservation protocols as demonstrated in recent works [66,67]. Figure 1 reports the stability index obtained for the Pickering emulsions based on the three different clays as functions of clay concentration. It should be noted that Lap above 1 mass% did not allow us to obtain a homogeneous dispersion and, therefore, a limit in the stabilizing ability of this nanoclay was evidenced. On the other hand, the most effective clay in oil stabilization was Kao that at 4 mass% allowed total stabilization of the oil. Sep clay can be considered a good dispersing agent, although its efficacy could not exceed 85% of oil stabilization. On the basis of these results, we considered a Pickering emulsion with an essential oil/water 1:3 mass ratio stabilized with Kao at 4 mass% to which, based on gel formation ability of laponite ® clay [68], the latter was added as thickener agent (4%). A milk-like gel dispersion was obtained, which appeared suitable for applications in surface cleaning (Figure 2).

Application
The chosen emulsion was tested (30 March 2019, temperature 14 • C, humidity 64%) on three outdoor surfaces (ceramic, marble, cement grit), attacked by biological agents (green algae Clorophyceae and cyanobacteria) by applying it on their surfaces and covering with a polyethylene film, for some hours (Figure 3). For ceramic surface (Figure 3a), the laying times were 1, 5, 10, and 24 h, after which the emulsion was removed with pads and water. Three tests were applied on the cement grit (1, 5, 24 h laying time) (Figure 3b). Then, the first one was removed by mechanical action, and the other two by washing with water. The emulsion was also tested on an outdoor marble surface subjected to atmospheric agents and presenting a biological attack (Figure 3c). In this case, the emulsion was left for 5 h and then removed with water and pads.
The results were very satisfying for all the tested materials, as shown in Figure 4, and the treated surfaces looked clean and devoid of biodeteriogens. After four months, the surfaces are still clean and free of the biodeteriogens.

Essential Oil Distillation
The fresh samples were ground in a Waring blender and then subjected to hydrodistillation for 3 h, according to the standard procedure previously described [69]. The oils were dried over anhydrous sodium sulphate and then stored in sealed vials, at −20 • C, ready for the gas chromatography-mass spectrometry (GC/MS) analysis. The sample yielded 0.35% (w/w) of oil.

Qualitative and Quantitative Analyses of Essential Oil (GS and GC/MS)
GC/MS analysis was performed on an Agilent Technologies 6850 N gas chromatograph coupled to a mass spectrometer (Agilent Technologies 5973) using a HP-5 MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.25 µm film thickness; J & W Scientific, Folsom) capillary column. The oven temperature programme was as follows: 5 min at 60 • C, subsequently 4 • C/min up to 220 • C, then 11 • C/min up to 280 • C, held for 15 min, for a total run of 67.29 min. Injector and detector temperatures were 280 and 300 • C, respectively. Helium (He) was used as the carrier gas, at a flow rate of 1 mL/min. Split ratio, 1:50; acquisition mass range, m/z 29-400. All mass spectra were acquired in electron-impact (EI) mode with an ionization voltage of 70 eV. Oil samples were diluted to 1:100 in n-hexane, and the volume injected was 2 µL. For 23 out of 28 essential oil constituents (Table 1), the identification consisted in the comparison of peak retention time, retention index, and mass spectrum with that of authentic standards (Sigma-Aldrich, Milan, Italy). Otherwise, the peak identification was based on the combination of linear retention indices and mass spectra with those reported in the WILEY275, NIST 17, ADAMS, and FFNSC2 libraries. Quantification of essential oil components was performed by peak-area normalization by considering an equal response factor for the different chemical classes.

Pickering Emulsion
In this work, Pickering emulsion was used as support to enable T. capitata essential oil application on stones surfaces. Water, oil, and clays were mixed to make the emulsion. For all Pickering emulsions, the mass percentage of clays was systematically changed, while the oil/water ratio was fixed at 1:3. A literature protocol was used to quantify the emulsion stability [59]. Briefly, the emulsions were sonicated (Transsonic 310/h) for 5 min, and then centrifuged at 1000 rpm (Heraeus Multifuge 3S+ centrifuge Thermo scientific). The quantity of separated oil phase was determined from optical photos after proper calibrations using ImageJ software. The stability index was calculated in terms of oil percentage incorporated into the emulsion and that did not show separation after the centrifugation protocol.

Conclusions
The Pickering emulsion of the essential oil of T. capitata is a typical emulsion that is called "lean emulsion" in the field of restoration of cultural assets. This emulsion is characterized by the presence of two immiscible products: an apolar substance (oil or mix of hydrocarbons) and a more polar one (water). Usually, a surfactant is added to emulsify these products together. In our case, the Pickering emulsion was characterized by the absence of surfactants and by the presence of a clay that acts as an emulsifier (kaolin) and another gelling agent (laponite). When the T. capitata oil Pickering emulsion is applied to a biologically degraded stone surface, an interaction is created between water, oil, stone surface, algae or bacteria. The biocidal phenolic components of the oil (carvacrol, thymol) act on and damage the plasma membrane of the cells, leading to biodeteriogens' death. Furthermore, this emulsion shows a cleaning power, thanks to the presence of water. Once the emulsion has been removed, dead biodeteriogens are also detached. Months later, it was observed that the biocidal action of the essential oil of T. capitata is still ongoing. The parts treated with the emulsion are still free of biological degradation and optically clean. There are currently no contraindications to the use of this essential oil alone or encapsulated in emulsions and polysaccharides. These are totally natural products that intervene microscopically at the cellular level, and, unlike the current quaternary ammonium salt chemicals, are not dangerous for the environment and the operator. The use of natural and ecological products in the field of restoration is growing rapidly and this product is harmless to the cultural heritage, to the operator and to the general environment. The only downside, if it is, is the typical smell of the oil, which persists for several days.