Potential cosmeceutical lamellar liquid crystals containing black longan (Dimocarpus longan Lour.) seed extract for MMP-1 and hyaluronidase inhibition

The aims of this study were to evaluate the biological activities of black Dimocarpus longan Lour. seed extracts and develop a lamellar liquid crystal (LLC). Different solvents, including petroleum ether, ethyl acetate, and 95% v/v ethanol, were used in the maceration of black D. longan seeds. The inhibitory activities on matrix metalloproteinase-1 (MMP-1) and hyaluronidase were evaluated. The irritating potency of D. longan seed extracts was determined using the hen's egg chorioallantoic membrane test. The extract with the strongest anti-ageing activities and no irritant impact was examined for its chemical contents using high-performance liquid chromatography and incorporated into the LLC. Various factors affecting the LLC formulations, including surfactant types and amounts, thickening agent types and amounts, and various oil types, were investigated. The results demonstrated that the ethyl acetate extract (EtOAc) was the most potent inhibitor of MMP-1 (IC50 = 21.7 ± 5.4 µg/mL) and hyaluronidase (oleanolic acid equivalent = 0.44 ± 0.03 g per g extract). Interestingly, its MMP-1 inhibition was equivalent to that of oleanolic acid, corilagin, and gallic acid. Furthermore, its hyaluronidase inhibition was equivalent to that of gallic acid and ellagic acid. Gallic acid, which was the most abundant compound (15.6% ± 0.06% w/w) was suggested as the compound responsible for the biological activities of EtOAc extract. Considering its potential anti-skin ageing properties with no irritation of EtOAc extract, it was incorporated into the most suitable LLC, which was composed of 5% w/w Lexfeel® D5 oil, 0.5% w/w Carbopol® U21, 2% w/w Liquid Crystal Cream Maker, and 92.5% w/w DI water. The LLC containing EtOAc extract presented birefringence under a polarizing light microscope, indicating its crystallinity. The formulation had good stability after heating–cooling cycles in terms of external appearance, crystallinity, viscosity, and pH (5.5). As a result, it is recommended as a potential cosmeceutical formulation for anti-skin wrinkling. It is proposed that more research be conducted on the safety and efficacy of the treatment on human volunteers.

www.nature.com/scientificreports/ demonstrated in Figure 1, all extracts and compounds displayed a dose-response relationship. Among different black D. longan seed extracts, EtOAc extract exhibited the most significantly potent MMP-1 inhibition with the lowest IC 50 value (p <0.05), followed by PET and EtOH extract. Interestingly, EtOAc extract was found to be as potent as corilagin, gallic acid, and oleanolic acid. However, ellagic acid, which is a gallic acid derivative, had no effect on the MMP-1 activity. The most plausible explanation could be due to the steric effect of ellagic acid when compared to gallic acid 21 and the additional functional group that was incongruent with the active site of MMP-1. MMPs, which are zinc-dependent endopeptidases that degrade ECM components and play a role in dermal turnover and remodeling, are expressed at very low levels in normal skin and remain inactive due to endogenous inhibitors 22 . However, during the inflammation process, exposure to UV irradiation, and even the natural ageing process, MMPs can be activated, resulting in enhanced ECM breakdown and skin wrinkles 23,24 . Among several types of MMPs, MMP-1 (interstitial collagenase) is the most closely associated with skin wrinkles as it breaks down the extracellular fibers composed of type I and III collagen 25 . Therefore, EtOAc extract can be suggested for further use as a cosmeceutical active ingredient for anti-skin wrinkling since it is a potent MMP-1 inhibitor.
Hyaluronidase-inhibitory activity of black D. longan seed extracts. The hyaluronidase-inhibitory activity of black D. longan seed extracts is shown in Figure 2. As oleanolic acid has long been known as a natural cosmeceutical component with various skin beneficial effects, particularly hyaluronidase-inhibitory activity 26 , the results of hyaluronidase inhibition were given in terms of oleanolic acid equivalent (OAE) in the present study. Gallic acid, ellagic acid, and corilagin, reported as the major bioactive components of black D. longan, were also investigated for their inhibitory effect on hyaluronidase 10 . Among black D. longan seed extracts, EtOAc extract was highlighted as the most potent hyaluronidase inhibitor with an OAE of 0.44 ± 0.03 g of oleanolic acid  www.nature.com/scientificreports/ per 1 g sample (p < 0.05). PET and EtOH extract inhibited hyaluronidase in a comparable manner with OAEs of 0.21 ± 0.02 and 0.17 ± 0.04 g of oleanolic acid per 1 g sample, respectively. Hyaluronidase is an enzyme related to skin wrinkle formation considering its key role in the breakdown of hyaluronic acid or hyaluronan, a major component of the three-dimensional network of skin ECM with crucial physiological functions in maintaining the skin structure and retaining water molecules in the skin 27 . Therefore, the anti-skin ageing effects could be attributed to EtOAc extract, which exerted a potent inhibitory effect on hyaluronidase,.
Irritation effect of black D. longan seed extracts. The fertilized hen's egg test on the chorioallantoic membrane (HET-CAM) 28,29 was used to determine the irritation effect of black D. longan seed extracts. This experiment is a well-known and trustworthy irritation study. Because the age of the animal's embryo was less than half of the incubation period, ethical approval was not required. Prior to the experiment, the HET-CAM test was validated using normal saline solution (NSS) as a negative control, whereas sodium lauryl sulfate (SLS) aqueous solution (1% w/v) was used as a positive control because of its irritating effects on the skin, resulting in increased transepidermal water loss (TEWL) and skin erythema 30 . The results shown in Figure 2 point out that NSS induced no irritation since the CAM showed no such signs, resulting in an irritation score (IS) of 0.0 ± 0.0. In contrast, SLS induced severe irritation with an IS of 10.4 ± 0.8. Hemorrhage, vascular lysis, and vascular coagulation were observed in the CAM after 5 min of exposure to SLS, and further severity can be detected after 1 h. Some small vessels disappeared after 1 h of exposure. Considering the results using NSS and SLS, the HET-CAM test was well validated since the negative control induced no irritation, whereas the positive control induced severe irritation. According to the validated HET-CAM assay, black D. longan seed extracts were safe and induced no irritation signs on the CAM after 5 min of exposure, as shown in Figure 3. The IS of all black D. longan seed extracts was 0.0 ± 0.0, classified as no irritation. Furthermore, irritation signs were not detected after 1 h of exposure. The results were comparable to those of NSS, the negative control. Since the HET-CAM test has been proposed as a useful in vitro assay for the irritation assessment of cosmetic formulations and ingredients, especially eye irritation 28 , it can be concluded that all black D. longan seed extracts are safe for topical application as an active cosmetic ingredient. Because the extracts from black D. longan seed, particularly EtOAc extract, demonstrated substantial anti-ageing activities through MMP-1 and hyaluronidase inhibition while producing no irritation, they can be considered safe for further cosmetic or cosmeceutical application.
Phenolic content of black D. longan seed extracts. Regarding EtOAc extract's significant MMP-1and hyaluronidase-inhibitory effects, it is an appealing natural anti-ageing active component for future cosmetic or cosmeceutical product development. As a result, high-performance liquid chromatography (HPLC) was used to analyze the chemical contents of EtOAc extract. The HPLC chromatogram of EtOAc extract (Fig-Figure 3. Chorioallantoic membrane of hen's egg after exposure to normal saline solution (NSS), 1% w/v sodium lauryl sulfate (SLS), black D. longan seed petroleum ether extract (PET extract), black D. longan seed ethyl acetate extract (EtOAc extract), and black D. longan seed ethanolic extract (EtOH extract). www.nature.com/scientificreports/ ure 4) revealed gallic acid as the most abundant compound with the amount of 15.6% ± 0.06% w/w. Moreover, corilagin and ellagic acid were only detected in small amounts of 2.2% ± 0.03% w/w and 2.6% ± 0.02% w/w, respectively. Apart from our previous study 10 , the chemical compositions of black D. longan seeds have rarely been investigated. This study's findings, which identified gallic acid as the predominant component of black D. longan seeds extracted using ethyl acetate, were consistent with our earlier research on D. longan seed ethanolic extract 10 . Corilagin and gallic acid were the compounds responsible for MMP-1-inhibitory activity of black D. longan seed extracts as they exerted potent MMP-1 inhibition with a comparable IC 50 value to oleanolic acid (Table 1), which is a well-known MMP-1 inhibitor. On the other hand, gallic acid and ellagic acid could be the bioactive compounds responsible for hyaluronidase inhibition since they possessed potent OAEs of 0.52 ± 0.01 and 0.53 ± 0.01 g of oleanolic acid per 1 g sample, respectively ( Figure 2). Therefore, gallic acid, corilagin, and ellagic acid are suggested for use as bioactive markers in further quantitative analysis of black D. longan seed extracts.

Lamellar liquid crystals (LLCs). Various factors affecting the LLC formation, including types and amounts
of surfactants, thickening agents, or oils, were investigated in the current study.
Effect of surfactant types and amounts. LLCs were developed using two different emulsifying systems, C20-22 alkyl phosphate and C20-22 fatty alcohol (Runemul TM Favor LC:LC), and sorbitan stearate and sucrose cocoate (Liquid Crystal Cream Maker: LCM). The physical appearances of LLCs slightly differed depending on the concentrations of LC and LCM in the formulations. Most formulations were opaque homogeneous semisolid, except for the formulation composed of 1% w/w surfactant. Therefore, it could be concluded that 1% w/w LC or LCM was not enough to produce a stable LLC formulation. The minimum amount of surfactant (LC and LCM) required for LLC preparation was 2% w/w. LCM could produce more pronounced birefringence under the polarizing light microscope than LC, as shown in Figure 5. Since birefringence represents crystallinity and is used to identify LLC formations 31 , LCM is suggested as the effective surfactant for the LLC formation. Furthermore, a higher concentration of each surfactant enhanced the crystallinity of the formulations. Although the viscosity of each formulation (Table 2) revealed that a higher amount of the surfactant led to higher viscosity, the viscosity of the formulations was not related to their crystallinity. LC could produce a formulation with higher viscosity than LCM at the same concentration. A previous study reported that the rheological properties of LLC formulation constituted their crystallinity and complexity 32 . The likely explanation for the results in this study might be due to the difference in the formulation type, whereby LCM produced LLCs but LC produced conventional emulsions. However, all formulations were stable after heating-cooling cycles because their viscosity and birefringence remained unchanged. In brief, 2% w/w LCM was selected for further study because it could produce LLC formulations with stable crystallinity, and only the minimum concentration was used.
Effect of thickening agent types and amounts. Since the incorporation of gelator molecules or so-called thickening agents into the lyotropic system resulted in a higher translational order for the gelled lamellar phases in comparison with gelator-free systems 33 , a variety of thickening agents, including xanthan gum, Carbopol ® U21, and sodium carboxymethylcellulose (SCMC), were added to the LLC formulations of 2% w/w LCM. Each formulation had a homogeneous opaque semisolid appearance with birefringence detected under the polarized light optical microscope ( Figure 6). Higher concentrations of thickening agents led to more pronounced birefringence, which was well related to their viscosity (Table 3). Xanthan gum and Carbopol ® U21 presented the same pattern of birefringence, whereas SCMC presented a small and widely dispersed pattern of birefringence. At the same concentration, the formulation of Carbopol ® U21 had a lower viscosity than that of xanthan gum. However, a large amount of SCMC was required for the same viscosity as xanthan gum. Regarding the natural color of xanthan gum, the formulation containing xanthan gum was pale yellow except at the concentration of 0.1% w/w, at which point the formulation was white. The xanthan gum LLC became a more intense yellow color upon increasing the xanthan gum concentration. In contrast, the formulation composed of Carbopol ® U21 was www.nature.com/scientificreports/ white, while that composed of SCMC was pale yellow without a color change upon increasing the concentration of the thickening agent. All LLC formulations remained stable after heating-cooling cycles in terms of external appearance and crystallinity. However, the viscosity of LLCs containing a low concentration of xanthan gum (0.1% w/w) and SCMC (0.5% w/w) was significantly enhanced after the stability test. The likely explanation might be due to the full hydration of the thickening agents after long-term storage. Xanthan gum, an exocellular biopolysaccharide produced by Xanthomonas campestris, has been reported to produce a gel-like structure at concentrations of 0.5% and above, while a gel-like structure is absent at 0.25% and below 34 . The results are in good agreement with the present study since the birefringence of the LLC formulation with 0.1% w/w xanthan gum (Fig. 6A) was the same as that of the LLC formulation without any thickening agent (Fig. 5E). However, the addition of xanthan gum to the LLC formulation resulted in higher viscosity since even a tiny quantity of polysaccharide can considerably enhance the viscosity of either water or the emulsion system 35 . However, the microstructure of xanthan gum is thermally unstable, i.e., sensitive to temperature changes with poor temperature resistance, because heating resulted in a progressive degradation of its network structure 36 . On the other hand, the viscosity of LLCs with a low concentration of SCMC, a semisynthetic anionic cellulose ether, increased dramatically after the stability test. The likely explanation might be due to the high sorption and great hygroscopicity of cellulose derivatives, as well as the structural changes in the native state upon changing temperature 37,38 . In addition to the unstable viscosity after heating-cooling cycles of LLCs from SCMC and xanthan gum, the yellow color was a limitation of using SCMC, while the sticky feeling after application was a limitation of using xanthan gum. Therefore, Carbopol® U21 is suggested as a suitable thickening agent in the LLC formulation for further studies.
Effect of various oil types. Various lipophilic components have been successfully used for LLC development, such as medium-chain triglycerides 39 , alkanes 40 , and edible oils 41 . A variety of oils, both synthesized and natural, were used in the liquid crystal development, including S. chinensis oil, P. dulcis oil, P. ocymoides seed oil, C. oleifera seed oil, mineral oil, and Lexfeel ® D5 oil. The addition of oils to the formulation resulted in increased crystal-  www.nature.com/scientificreports/  www.nature.com/scientificreports/ linity ( Figure 7) and viscosity (Table 4). Different oils could generate LLC formulations with different texture, odor, and color. However, the same concentration (5% w/w) of each oil had no effect on the viscosity. Lexfeel ® D5 oil, a mixture of synthesized neopentyl glycol diheptanoate and isododecane, was selected for further study since it produced LLCs that were light, dry, odorless, and colorless.

Lamellar liquid crystals containing D. longan seed extracts.
Considering the most potent MMP-1-inhibitory activity equivalent to oleanolic acid, corilagin, and gallic acid, as well as the most potent hyaluronidase-inhibitory activity equivalent to gallic acid and ellagic acid, EtOAc extract was selected as a natural active cosmeceutical ingredient for anti-ageing. However, to achieve inhibition against MMP-1 and hyaluronidase, the active compounds must be delivered to the dermis layer, which is the target site of action. An LLC, containing 5% w/w Lexfeel ® D5 oil, 0.5% w/w Carbopol ® U21, 2% w/w LCM, and 92.5% w/w DI water, was developed for delivering EtOAc extract into the skin layer. As only a proportion of EtOAc extract in the LLC formulation can be delivered to the target site (dermis layer), the concentration of EtOAc extract employed in the formulation was made as high as practicable. The concentration of EtOAc extract in the LLC formulation was 0.5% w/w, with 250 times the IC 50 value against MMP-1. At this concentration, EtOAc extract caused no irritation. Furthermore, there was no problem with the dissolution of EtOAc extract in the LLC formulation. The LLC formulation containing D. longan seed extract (LLC-EtOAc) was a pale-yellow semisolid ( Figure 8A) with characteristic odor. Birefringence ( Figure 8B) indicated the liquid crystal nature of the formulation. After incorporation of EtOAc  www.nature.com/scientificreports/ extract in the LLC formulation, viscosity increased from 1.83 ± 0.22 to 2.07 ± 0.06 mPa·s (p < 0.05). The formulation exhibited good stability after heating-cooling cycles in terms of external appearance, crystallinity, viscosity, and pH (5.5). The external appearance of the formulation remained unchanged in texture, odor, and color ( Figure 8C). Furthermore, the birefringence was also the same ( Figure 8D). The viscosity of the LLC-EtOAc after the accelerated stability study was 2.22 ± 0.08 mPa·s, which was not different from the initial viscosity (p > 0.05). These findings highlight LLC-EtOAc as a stable formulation. However, further analysis of the bioactive compounds in the formulation using the stability test is proposed.

Conclusions
Black D. longan seed extracts, particularly EtOAc extract, have the potential to be used as bioactive cosmetic ingredients. According to the most potent inhibitory activities toward MMP-1 and hyaluronidase, EtOAc extract is of interest to be further investigated for its phytochemical constituents and safety profile. Gallic acid was identified as the most abundant compound (15.6% ± 0.06% w/w) responsible for the biological activities of EtOAc extract. The inhibition of both MMP-1 and hyaluronidase by EtOAc extract was equivalent to gallic acid. The irritation effect of black D. longan seed extracts, investigated by the HET-CAM test, revealed their safety as none of the extracts induced any irritation signs on the CAM. Considering its potential anti-skin ageing properties with no irritation, EtOAc extract was incorporated into an LLC, and it is recommended as a potential cosmeceutical formulation for anti-skin wrinkling via MMP-1-and hyaluronidase-inhibitory activities. The findings from this research encourage the utilization of black D. longan seeds, which would not only reduce the waste product but also enhance the economic value of D. longan by processing it into a natural anti-ageing cosmetic ingredient and finally into cosmetic products. However, prior to launching skincare products containing black D. longan seed extracts, further studies on human volunteers evaluating the safety and efficacy of the LLC containing EtOAc extract are suggested. The bioactive compounds in the formulation should also be investigated, along with a stability study to determine the shelf-life of the product.  The weight ratio of black D. longan seed powder and solvent was 1:3. Each extraction was repeated for three cycles. After that, the extracting solvent was filtered and removed using a rotary evaporator (Buchi Labortechnik GmbH, Essen, Germany). All extracts were stored in well-closed containers in the refrigerator (4-6 ºC) until further use.

MMP-1-inhibitory activity determination of black D. longan seed extracts. The MMP-1-inhib-
itory activity of each D. longan seed extract was investigated by spectrophotometric methods 43,44 . Various concentrations of the samples, ranging from 0.0125 to 0.8 mg/ml, were investigated for their MMP-1-inhibitory activities. Briefly, 0.5 units/mL of MMP-1 from Clostridium histolyticum was mixed with the test solution, followed by a substrate (FALGPA). Immediately, the whole mixture was kinetically measured for the absorbance at 340 nm for 20 min using a DTX880 multimode detector (Beckman Coulter, Indianapolis, IN, USA). The inhibitory activity of each sample was calculated using the following equation: % inhibition = (1 − A/B) × 100, where A is the absorbance of the mixture with the test solution and B is the absorbance of the mixture without the test solution. Oleanolic acid was applied as a positive control. The experiment was carried out three times.
Hyaluronidase-inhibitory activity determination of black D. longan seed extracts. The hyaluronidase inhibitory activity of each D. longan seed extract was investigated by spectrophotometric methods 43,44 . The sample concentration of 0.1 mg/mL was investigated for its hyaluronidase-inhibitory activity. Prior to the experiment, the enzyme activity of hyaluronidase was assessed, and a level of more than 90% enzyme activity was used for further anti-hyaluronidase activity determination. Briefly, 15 units of bovine testis-derived hyaluronidase was mixed with the test solution. After incubation at 37 °C for 10 min, hyaluronic acid was added and incubated for another 45 min. The whole mixture was measured for the absorbance at 600 nm using a DTX880 multimode detector (Beckman Coulter, Indianapolis, IN, USA). The inhibitory activity of each sample was reported as oleanolic acid equivalent on hyaluronidase inhibition. The experiment was carried out three times.

Irritation test of black D. longan seed extracts by hen's egg test chorioallantoic membrane (HET-CAM) assay.
The irritation of black D. longan seed extracts was evaluated by the HET-CAM test 29 .
The experiment is a well-known and trustworthy irritation study. Because the age of the animal's embryo was less than half of the incubation period, ethical approval was not required. The fertilized hen eggs were incubated for 7 days at 37.5 ± 0.5° C with 55% ± 7% relative humidity. After opening the air chamber above the eggshell using a rotating cutting blade, the inner membrane was gradually removed. Then, 30 μL of 5 mg/ml sample solutions were dropped onto the chorioallantoic membrane (CAM). The irritation signs on CAM were immediately observed under a stereomicroscope (Olympus, Tokyo, Japan) and continuously observed for 5 min. www.nature.com/scientificreports/ of 1% w/v SLS was applied as a positive control, and normal saline solution (0.9% w/v NaCl) was applied as a negative control. The experiment was carried out twice.
Determination of phytochemical constituents of black D. longan seed extracts using high-performance liquid chromatography (HPLC). The amounts of gallica acid, corilagin, and ellagic acid were evaluated using an HP 1100 chromatographic system (Hewlett-Packard, Waldbronn, Germany) 6 . A 0.45 mm millipore filter, type GV (Millipore, Bedford, MA) was used for the filtration of both the test solution and the mobile phase. A gradient mobile phase system, consisting of 0.05% formic acid in acetonitrile (A) and 0.05% formic acid aqueous solution (B), was programmed for gradient elution through a Eurospher II 100-5 C18 column (250 × 4.6 mm, i.d. 5 µm, Knauer, Berlin, Germany) as follows: 10% A (0-8 min), 20% A (8-28 min), 30% A (28-30 min), and 10% A (30-35 min). Each D. longan seed extract was dissolved in acetonitrile, and the sample solution was eluted at a flow rate of 1.0 mL/min. The content of ellagic acid was calculated using the area under the curve of each peak detected by a UV detector set at 280 nm.

Effects of surfactant types and concentrations.
To investigate the effects of types and concentrations of surfactants, various formulations of LLC were developed, with the components shown in Table 5. Each liquid crystal system was prepared using the conventional beaker method. Concisely, an aqueous phase (deionized water and moisturizing agents) was mixed and heated until 75 °C in a glass beaker. Simultaneously, an oil phase was heated until 70 °C in another glass beaker. The oil phase was gradually introduced into an aqueous phase and stirred using a stirring rod while cooling to room temperature. Each formulation was kept in a well-closed container at room temperature until the next experiment.

Effects of thickening agent types and concentrations.
To investigate the effects of types and concentrations of thickening agents, various formulations of LLC were developed, with the components shown in Table 6. Each thickening agent was previously dispersed in an aqueous phase. Then, the gelled aqueous phase was heated and used for the LLC preparation using the beaker method as described above. When using Carbopol ® Ultrez 21 as the thickening agent, triethanolamine was used as a neutralizing agent. The pH of the formulation  www.nature.com/scientificreports/ was adjusted by adding triethanolamine in the final step of preparation. Each formulation was kept in a wellclosed container at room temperature until the next experiment.
Effects of oil phase. To investigate the effects of oil type, various formulations of LLC were developed, with the components shown in Table 7. Each liquid crystal system was prepared using the conventional beaker method as previously described. Each formulation was kept in a well-closed container at room temperature until the next experiment.

Effects of oil concentration.
To investigate the effects of oil concentration, various formulations of LLC were developed, with the components shown in Table 8. Each liquid crystal system was prepared using the conventional beaker method as previously described. Each formulation was kept in a well-closed container at room temperature until the next experiment. Development of LLC containing D. longan seed extracts. The D. longan seed extract, which exhibited the highest anti-skin wrinkling activity with no irritation effect, was selected for use as an active cosmetic or cosmeceutical ingredient in the LLC formulation. On the other hand, the LLC formulation, which exhibited good characteristics and stability after the heating-cooling test, was selected for the incorporation of selected D. longan seed extract. The formulation of the LLC containing the black D. longan seed extract is shown in Table 9.
In the preparation process of the LLC containing D. longan seed extracts, D. longan seed extract was finally  www.nature.com/scientificreports/ added to the preformulated LLC and mixed thoroughly. The LLC formulation containing D. longan seed extract was then characterized and tested for their stability via eight heating-cooling cycles.
Statistical analysis. All values were given as means ± standard deviation. The statistical analysis involved a t-test and ANOVA using SPSS software (SPSS Statistics 21.0, IBM Corporations, New York, NY, USA). A value of p < 0.05 was accepted as significant.

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.