**1. Introduction**

*Physalis alkekengi* L. (family Solanaceae), also known as the Chinese lantern, Japanese lantern, bladder cherry, winter cherry, and by many other common names, is a species indigenous to Asia and Southern Europe, further naturalized worldwide [1,2]. Nowadays,

**Citation:** Popova, V.; Petkova, Z.; Mazova, N.; Ivanova, T.; Petkova, N.; Stoyanova, M.; Stoyanova, A.; Ercisli, S.; Okcu, Z.; Skrovankova, S.; et al. Chemical Composition Assessment of Structural Parts (Seeds, Peel, Pulp) of *Physalis alkekengi* L. Fruits. *Molecules* **2022**, *27*, 5787. https:// doi.org/10.3390/molecules27185787

Academic Editor: Sabina Lachowicz-Wi´sniewska

Received: 9 August 2022 Accepted: 2 September 2022 Published: 7 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the Chinese lantern is encountered as cultivated and ornamental varieties or as a wildgrowing plant in various climatic zones, from Central and Southern Europe to South and Northeast Asia.

The species is the only one in the genus *Physalis*, which is found as wild populations in Bulgaria, growing in different regions at altitudes up to 1200–1500 m [3,4]. The local name of the species is 'mekhunka', and its preservation and use are regulated by the Medicinal Plants Act [5]. Survey data [6] have documented a steady export of about 760 kg fresh *P. alkekengi* fruit per year in the period 2001–2005. In Bulgaria, the Chinese lantern grows as a perennial plant with widely spreading roots and a slightly branched or unbranched stem with a height between 40 and 60 cm. The fruit ripen in August–September, presenting as small, oval, brightly colored berries containing numerous tiny seeds, and completely covered by the characteristic wide orange-red papery calyx (husk).

Despite its identification as a medicinal plant, there are practically no data from investigations of *P. alkekengi* phytochemical composition in Bulgaria, nor from studies of its biological activities or range of application. Ivanov et al. [7] reported that the fruits contain red pigments, physalin, citric, malic and tartaric acids, vitamin C, and bitter substances. The seeds alone can contain up to 25% of the oil [7]. According to numerous studies on the species in its natural areas of distribution, over 100 bioactive metabolites have been identified in the fruit and other aerial parts of the plant, including alkaloids, nucleosides, peptides, terpenoids, megastigmanes, aliphatic derivatives, organic acids, coumarins, sucrose esters, polysaccharides, and carotenoid derivatives [8–17].

The Chinese lantern has been recognized for centuries as a medicinal plant in the traditional medical practices of many countries, due to its anti-inflammatory, antibacterial, antiseptic, sedative, laxative, diuretic, hypoglycemic, spasmolytic, and other effects, as well as for the relief of malaria and syphilis symptoms [1,18,19]. In Chinese medicine, *P. alkekengi* (Physalis calyx seu Fructus) is a remedy for a number of diseases—from sore throat, eczemas, and rheumatism to hepatitis, urinary disorders, and tumors [1,2,10,14,18]. In turn, Bulgarian folk medicine recommends the use of fresh or dried fruit for the treatment of liver diseases, combining hepatitis and ascites [3]. Dried fruits are also used as a painkiller for kidney and bladder stones, inflammations of the urinary tract, and hemorrhoids. In topical application, fresh juice or whole fruits relieve skin irritations, wounds, and inflammation. A daily consumption of at least 10–15 fresh berries (or the equivalent 20 mL freshly squeezed juice) is highly recommended [3].

*P. alkekengi* is also recognized as a functional food, being a rich source of valuable nutrients—vitamins A and C, minerals, unsaturated fatty acids, phenolics, phytosterols, and pectic substances [18,20–22]. It should be noted that only fully ripened berries are suitable for consumption (unripe fruit and all aerial parts of the plant are toxic if swallowed), having a juicy texture, fresh flavor, and a slightly bitter taste, which normally disappears after fruit freezing.

There is relatively limited information about the cosmetic uses of *P. alkekengi* in the form of aqueous, ethanolic, and other extracts from the fruit and calyces, which are incorporated in different cosmetic formulations, taking advantage of its beneficial effects on the skin (protective, soothing, anti-ageing, anti-pigmentation, and other effects) [1,23]. Those and related investigations have supported the inclusion of *P. alkekengi* fruit and calyx extracts (CAS No 90082-67-0) in the Cosmetic Ingredient Database (CosIng) of the European Commission, in the category of cosmetic ingredients with skin conditioning functions [24].

To the best of our knowledge, there are not enough data on the distribution of phytonutrients and other chemical compounds among the structural parts of the fruit (peel, pulp, and seeds), nor on the characteristics of *P. alkekengi* growing in different regions of Bulgaria. The main objective of this study was therefore to provide a comparative assessment of the chemical composition of the structural parts of the fruit in two Bulgarian phenotypes of *Physalis alkekengi* L., thus supplementing the already existing knowledge about the species and expanding its possible prospective use in nutrition and cosmetics.

#### **2. Materials and Methods 2. Materials and Methods** *2.1. Plant Material*

#### *2.1. Plant Material* Two phenotypes of *Physalis alkekengi* found in Bulgaria were analyzed in this study.

cosmetics.

Two phenotypes of *Physalis alkekengi* found in Bulgaria were analyzed in this study. Fully ripe fruits (about 150 pieces for each phenotype) were collected in August—September 2020 from wild plant populations in Central Southern Bulgaria (PA-SB phenotype; the city of Plovdiv; 42◦1402600 N 24◦7002400 E), and North-Eastern Bulgaria (PA-NB phenotype; near Ivanski village, Shumen region; 43◦0702400 N 27◦0403500 E). Species identification was confirmed by the botanist at the Department of Botany, Plovdiv University, Bulgaria. Fully ripe fruits (about 150 pieces for each phenotype) were collected in August— September 2020 from wild plant populations in Central Southern Bulgaria (PA-SB phenotype; the city of Plovdiv; 42°14'26" N 24°70'24" E), and North-Eastern Bulgaria (PA-NB phenotype; near Ivanski village, Shumen region; 43°07'24" N 27°04'35" E). Species identification was confirmed by the botanist at the Department of Botany, Plovdiv University, Bulgaria.

regions of Bulgaria. The main objective of this study was therefore to provide a comparative assessment of the chemical composition of the structural parts of the fruit in two Bulgarian phenotypes of *Physalis alkekengi* L., thus supplementing the already existing knowledge about the species and expanding its possible prospective use in nutrition and

According to the objectives of the study, fresh fruits (Figure 1) were de-husked and carefully divided into structural parts (seeds, peel, and pulp), that were analyzed individually or in mixture due to determination requirements. According to the objectives of the study, fresh fruits (Figure 1) were de-husked and carefully divided into structural parts (seeds, peel, and pulp), that were analyzed individually or in mixture due to determination requirements.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 3 of 17

**Figure 1.** Fresh fruits of *P. alkekengi*: (**a**) PA-SB phenotype from Central Southern Bulgaria; (**b**) PA-NB phenotype from North-Eastern Bulgaria. Photos by authors. **Figure 1.** Fresh fruits of *P. alkekengi*: (**a**) PA-SB phenotype from Central Southern Bulgaria; (**b**) PA-NB phenotype from North-Eastern Bulgaria. Photos by authors.

#### *2.2. Basic Evaluation of Fresh Berries and Their Structural Parts 2.2. Basic Evaluation of Fresh Berries and Their Structural Parts*

The proportion (%) of the structural fruit parts (peel, pulp, and seeds) in fresh berries was obtained by gravimetrical determination of each element's fresh weight (fw) (Mettler-Toledo, Switzerland; precision ± 0.0001 g) for 100 randomly selected fruits. Seed absolute weight (g) was obtained as mean by weighing of 1000 seeds on a precision balance (Mettler-Toledo, Switzerland; precision ± 0.0001 g). The proportion (%) of the structural fruit parts (peel, pulp, and seeds) in fresh berries was obtained by gravimetrical determination of each element's fresh weight (fw) (Mettler-Toledo, Switzerland; precision ± 0.0001 g) for 100 randomly selected fruits. Seed absolute weight (g) was obtained as mean by weighing of 1000 seeds on a precision balance (Mettler-Toledo, Switzerland; precision ± 0.0001 g).

The moisture content of each fruit sample in the study (seeds, peel, and pulp) was determined by drying in a laboratory drying oven (Robotika, Velingrad, Bulgaria) at 103 ± 2 °C to the constant weight [25]. The results from the chemical analyses in the study were further re-calculated and presented on a dry weight (dw) basis. The moisture content of each fruit sample in the study (seeds, peel, and pulp) was determined by drying in a laboratory drying oven (Robotika, Velingrad, Bulgaria) at 103 ± 2 ◦C to the constant weight [25]. The results from the chemical analyses in the study were further re-calculated and presented on a dry weight (dw) basis.

In the first step of chemical analysis, the samples were analyzed in two structural forms, as seed samples and peel/pulp samples. In the first step of chemical analysis, the samples were analyzed in two structural forms, as seed samples and peel/pulp samples.

Cellulose content (crude fiber) in the seed and peel/pulp samples was determined by a slight modification of a method described earlier [26]. The hydrolysis of the plant material (1.0 g) was carried out with 16.5 mL 80% CH3COOH and 1.5 mL concentrated HNO3 for 1.5 h at 100 °C. The filtrated residue was dried at 103 ± 2 °C for 24 h, cooled in a dessicator, and weighed for the quantitative determination of cellulose. The results are presented on a dry weight basis. Cellulose content (crude fiber) in the seed and peel/pulp samples was determined by a slight modification of a method described earlier [26]. The hydrolysis of the plant material (1.0 g) was carried out with 16.5 mL 80% CH3COOH and 1.5 mL concentrated HNO3 for 1.5 h at 100 ◦C. The filtrated residue was dried at 103 ± 2 ◦C for 24 h, cooled in a dessicator, and weighed for the quantitative determination of cellulose. The results are presented on a dry weight basis.

Protein content in the seed and peel/pulp fractions was analyzed by the Kjeldahl method [25] using an UDK 152 unit (Velp Scientifica Srl, Usmate Velate, Italy). The Protein content in the seed and peel/pulp fractions was analyzed by the Kjeldahl method [25] using an UDK 152 unit (Velp Scientifica Srl, Usmate Velate, Italy). The conversion to protein content of the determined nitrogen content, present as ammonia in the digested sample, was by the multiplication factor of 6.25.

Oil content in the seed and in the peel/pulp parts was determined after extraction with *n*-hexane (Soxhlet, for 8 h), followed by evaporation of the solvent on a rotary vacuum evaporator (at 40 ◦C) and under a stream of nitrogen [27].

#### *2.3. Determination of Fatty Acids in Seed and Peel Oils*

In the second step of the analysis, the oils from the fruit seeds and peel were isolated separately and then analyzed for the composition and content of the fatty acids and tocopherols. The oil fractions from seeds and peel were obtained by extraction with *n*-hexane, as described above [27].

Fatty acids in the extracted seed and peel oils were determined after transmethylation with 2% H2SO4 in CH3OH at 50 ◦C [28,29]. The GC analysis was performed on a Hewlett Packard 5890A unit, with a flame ionization detector (FID) (Santa Clara, CA, USA) and a capillary Supelco 2560 column, 75 m × 0.25 mm × 18 µm (i.d.). The column temperature increase was from 130 ◦C (4 min) to 240 ◦C (5 min) at 15 ◦C/min; injector/detector temperatures were 250 ◦C; the flow rate of the carrier gas (hydrogen) was 0.8 mL/min; the split was 50:1. Fatty acid identification was completed by referring to the retention times of fatty acid methyl esters (FAME) in a standard mixture of 37 components (Supelco, Bellefonte, PA, USA).

#### *2.4. Determination of Tocopherols in Seed and Peel Oils*

Tocopherols were analyzed directly in the seed and peel oils, without saponification, by HPLC method. A 2% solution of the respective oil in *n*-hexane was prepared, and 20 µL was injected in the Merck-Hitachi unit (Merck, Darmstadt, Germany); column 250 mm × 4 mm Nucleosil Si 50-5; fluorescent detector Merck-Hitachi F-1000 (Merck, Darmstadt, Germany). The flow rate of the mobile phase (*n*-hexane: dioxan 96:4, *v*/*v*) was 1.0 mL/min; detector excitation was at 295 nm, emission at 330 nm. Tocopherol identification was based on comparison with reference standards (DL-*α*-, DL-β-, DL-*γ*- and DL-*δ*-tocopherols, 98% purity) [30].

#### *2.5. Determination of Amino Acids in Seed Cakes*

In the third step, the seed cakes remaining after seed oil extraction were analyzed for the composition and content of amino acids and mineral elements.

Amino acids were detemined after hydrolysis of the seed cake material and completed with 6 N HCl at 105 ◦C for 24 h in sealed glass ampules. Ampule content was then evaporated at 40–50 ◦C under vacuum, and the residue was dissolved in 20 mM HCl and filtered. The free amino acids resulting from protein hydrolysis were derivatized using the AccQ-Fluor kit, WATO52880 (Waters Corporation, Milford, MA, USA). The HPLC separation of the resulting AccQ-Fluor amino acid derivatives was performed on an Elite LaChrome (Hitachi, Tokyo, Japan) unit, with a reverse phase C18 AccQ-Tag (3.9 mm × 150 mm) column (at 37 ◦C). WATO52890 buffer (Waters Corporation, Milford, MA, USA) and 60% acetonitrile were the eluting phases; the injected sample volume was 20 µL, heated to 55 ◦C. The unit was equipped with a diode array detector (DAD) (Hitachi, Tokyo, Japan), and the detection was performed at the wavelength 254 nm.

#### *2.6. Determination of Mineral Elements in Seed Cakes*

Mineral elements in the seed cakes were determined after mineralization at 450 ◦C; the resultant ash was first dissolved in concentrated HCl and then in 0.1 mol/L HNO3 [31]. The atomic absorption spectrometry (AAS) was performed on a Perkin Elmer/HGA 500 instrument (Norwalk, CT, USA). The detection wavelengths were: Na, 589.6 nm; K, 766.5 nm; Mg, 285.2 nm; Ca, 317.0 nm; Zn, 213.9 nm; Cu, 324.7 nm; Fe, 238.3 nm; Mn, 257.6 nm; Pb, 283.3 nm; Cd, 228.8 nm; Cr, 357.9 nm. The elemental identification was completed by comparison with standard metal salt solutions, and the estimation of metal ion concentration by using calibration curves (built for 1 µg/mL standard salt solutions).

#### *2.7. Determination of Volatiles in Fruit Concretes*

Finally, the fruit concretes were analyzed. These were obtained from *P. alkekengi* peel and pulp as concentrated aromatic products, which are commonly used in perfumery and cosmetics.

In the obtainment of fruit concretes, the samples were subjected to a double extraction with n-hexane, at a temperature of 40 ◦C and a solid-to-solvent ratio of 1:10 (*w*/*v*). The duration of the first and second extraction was 60 min and 30 min, respectively. The extracts were then combined and concentrated on a rotary vacuum evaporator until complete solvent removal at a temperature 40 ◦C. The yield of peel and pulp concretes was determined gravimetrically (%, *w*/*w*) and calculated on a dry weight basis. The initial moisture content of the extracted plant materials was as follows: peel—53.85 ± 0.45% (PA-SB) and 49.73 ± 0.41% (PA-NB); pulp—79.43 ± 0.62% (PA-SB) and 82.99 ± 0.71% (PA-NB). The color and appearance of the concretes were determined by visual assessment.

The GC analysis for the determination of the volatile composition of the obtained fruit concretes was performed on an Agilent 7890A instrument (Agilent Technologies Inc., Santa Clara, CA, USA), with the following parameters: HP-5ms column, 30 m × 250 mm × 0.25 µm (i.d.); oven temperature increased at a rate of 5 ◦C/min from 35 ◦C (3 min) to 250 ◦C (3 min), total run time 49 min; carrier gas (helium) at 1 mL/min constant rate; 30:1 split mode. The GC-MS analysis employed an Agilent 5975C inert XL EI/CI mass selective detector (MSD) (Agilent Technologies Inc., Santa Clara, CA, USA), under the same operational conditions as in the GC analysis. Mass spectra acquisition was at 70 eV in electron impact (EI) mode; MS scan was from 50 to 550 *m*/*z*. The ionization source temperature was 230 ◦C, and the MS quad and the injector temperatures were 150 ◦C and 250 ◦C, respectively. Mass spectra were read using the built-in toolkit of 2.64 AMDIS (Automated Mass Spectral Deconvolution and Identification System; NIST, Gaithersburg, MD, USA) software. The identification of volatiles was based on comparison of their retention (Kovats) indices (RI) and MS fragmentation patterns with spectral library data [32,33]. Components were listed in ascending order of the RI, calculated using a standard calibration mixture of *n*-alkanes (C8–C40) in hexane, under the same operational conditions. Compound concentrations were calculated as percentage of the total ion current (TIC), after normalization of the recorded peak areas.

#### *2.8. Statistics*

All measurements in the study were performed in triplicate (*n* = 3), except for the fruit parts proportion (*n* = 100). The results are presented as the mean value with the corresponding standard deviation (SD). ANOVA and Tukey multiple comparison test were used as statistical tools in the assessment of significant differences at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Basic Evaluation of Fruit Structural Parts*

The proportions of fruit structural parts (peel, seeds, and pulp, respectively) in the analyzed fresh fruits of two *P. alkekengi* phenotypes (denoted as PA-SB and PA-NB) are presented in Table 1. Fruit pulp accounted for about 70% of fresh fruit weight, while seeds constituted about a quarter of the fruit weight, with no significant deviations between the phenotypes. Seed dimensions varied, however, with seed absolute weight being 1.68 ± 0.01 g (mean of 1000 seeds results) for PA-SB phenotype, and 1.52 ± 0.01 g (per 1000 seeds) for PA-NB. The average number of seeds in a single berry was 195 ± 1.80 for PA-SB, and 107 ± 0.90 for PA-NB phenotype.

**Table 1.** Proportion of fruit structural parts of two *P. alkekengi* phenotypes (PA-SB and PA-NB).

Results: mean value ± standard deviation (*n* = 100). Different letters in the same row indicate significant differences (*p* < 0.05).

The basic macro component characteristics (cellulose, protein, oil) of the studied fruit fractions (the isolated seeds and the combined peel/pulp fraction) are presented in Table 2. The data indicate some differences between the phenotypes, with slightly higher oil amounts in PA-NB, and higher cellulose and protein amounts in the PA-SB phenotype. As seen from the data, the seeds were the primary site of oil accumulation in the fruit, although the combined peel/pulp samples also had detectable amounts of the oil fraction. The seeds of *P. alkekengi* were a sufficiently rich source of oil, yielding 14–17% oil, thus approximating the data for a related *Physalis* species, *P. peruviana* pomace oil (19.3%) detected by Ramadan [34], as well as those for soybean oil (18%) [35], grapeseed oil (8–20%) [36], or *P. alkekengi* seed oil [37].

**Table 2.** Macro component characteristics of fruit structural parts of two *P. alkekengi* phenotypes (PA-SB and PA-NB).

Results: mean value ± standard deviation (*n* = 3). Different letters in the same row indicate significant differences (*p* < 0.05).

Similarly, the seeds contained most of the fruit's protein and fiber. The cellulose content was several times higher in the seeds (6–8% dw) than in the peel/pulp residues (1.5–2% dw). The reported data were close to the cellulose contents detected in the whole fruits of different cape gooseberry (*P. peruviana*) phenotypes measured by Petkova et al. [38], as well as in different fruit and vegetable pomaces, such as apples and tomatoes [39].

#### *3.2. Determination of Fatty Acids in Seed and Peel Oils*

The significant oil yield from the seeds of *P. alkekengi* fruit was the reason for subjecting the extracted seed oil to a more detailed analysis in order to reveal its micro component characteristics.

As seen in Table 2, the combined peel/pulp fraction was also associated with the presence of oil fraction, although in a minor amount. The individual analysis of the two fruit parts constituting the combined sample revealed that oil content in the pulp was minimal (below 1% dw) in both phenotypes, while the peel contained 2.54 ± 0.02% and 2.05 ± 0.02% oil (dw) in the PA-NB and PA-SB phenotypes, respectively. Therefore, it was considered interesting to identify the composition of the peel fraction oil, as well, in view of a more complete insight into the composition of *P. alkekengi* fruits.

The results regarding the fatty acid composition of the seed and peel oils of the two phenotypes are presented in Table 3, and an example of the obtained FAME chromatograms is shown in Figure 2. The data proved significant variations in the number and distribution of the identified fatty acids in the seed and peel oils, while the differences between the phenotypes were less pronounced.

The fatty acid composition of *P. alkekengi* seed oil was dominated by unsaturated fatty acids in a ratio of about 7:1 to saturated ones; this was the same for both phenotypes. The ratio between polyunsaturated and monounsaturated fatty acids was also favorable and comparable for the phenotypes, being about 5:1. The proportions of unsaturated and saturated fatty acids, however, were reversed and much more unfavorable in the peel oils, being 32:68 in PA-SB and 44:56 in PA-NB, respectively.

The predominant fatty acid in the seed oils was linoleic acid (73.67% in PA-SB and 74.43% in PA-NB), followed by oleic and palmitic acids, while the major fatty acids in peel oils were palmitic (57.88% in PA-SB and 36.21% in PA-NB) and oleic (24.02% in PA-SB and 30.08% in PA-NB) acids.

**Table 3.** Fatty acid composition of seed and peel oils of two *P. alkekengi* phenotypes (PA-SB and PA-NB).

nd 1 = not detected; Results: mean value ± standard deviation (*n* = 3). Different letters in the same row indicate significant differences (*p* < 0.05).

Unlike the similar distribution of fatty acids in the seed oils, there were differences between the two phenotypes in the individual fatty acid composition of the oil derived from fruit peel alone, especially with regard to palmitic, palmitoleic, stearic, and some other acids. Although the results revealed a more favorable composition in the isolated seed oils, they supported the feasibility of oil extraction from *P. alkekengi* fruit, using both whole fruit and seeds alone. Regarding the individual fatty acid composition of the seed oils, our results differed only numerically from the data in the previous study [37], which identified linoleic (86.9%) and palmitic (6.6%) acids as the major fatty acids in the seed oil, at a ratio of unsaturated to saturated fatty acids of about 14:1. Our results were fully compliant with the data available for *P. peruviana* oils [34], which pointed out that pulp/peel oil was characterized by high amounts of saturated fatty acids, while *ω*-3 acids (α-linolenic) were found in lower levels.

#### *3.3. Determination of Tocopherols in Seed and Peel Oils*

The tocopherol composition of the extracted oils is presented in Table 4. As seen in the table, there was impressive differentiation in the content of the bioactive tocopherols on the bases of fruit structural parts and phenotype. Seed oils, despite the phenotype-related differences observed, contained considerably more tocopherols than peel oils. The data showed about a 2.5 times higher concentration of the biologically active tocopherols in the oil isolated from the seeds of PA-SB berries than for the PA-NB phenotype.

row indicate significant differences (*p* ˂ 0.05).

**Figure 2.** Chromatograms of the fatty acid composition of seed oils of two *P. alkekengi* phenotypes (PA-SB and PA-NB): a**—**capric acid; b**—**myristic acid; c**—**pentadecylic acid; d**—**palmitic acid; e palmitoleic acid; f**—**heptadecenoic acid; g**—**stearic acid; h**—**oleic acid; i**—**linoleic acid; j**—**linolenic acid; k**—**eicosadienoic acid; l**—**eicosatrienoic acid; m**—**eicosapentaenoic acid; n**—**eicosatetraenoic acid. **Figure 2.** Chromatograms of the fatty acid composition of seed oils of two *P. alkekengi* phenotypes (PA-SB and PA-NB): a—capric acid; b—myristic acid; c—pentadecylic acid; d—palmitic acid; e—palmitoleic acid; f—heptadecenoic acid; g—stearic acid; h—oleic acid; i—linoleic acid; j—linolenic acid; k—eicosadienoic acid; l—eicosatrienoic acid; m—eicosapentaenoic acid; n—eicosatetraenoic acid.

nd 1 = not detected; Results: mean value ± standard deviation (*n* = 3). Different letters in the same

*3.3. Determination of Tocopherols in Seed and Peel Oils*  The tocopherol composition of the extracted oils is presented in Table 4. As seen in **Table 4.** Tocopherol composition of seed and peel oils of two *P. alkekengi* phenotypes (PA-SB and PA-NB).

(28.44% and 23.42% of the total content, PA-SB and PA-NB, respectively). The peel oils showed completely different tocopherol profiles**—***γ*-tocopherol was dominating in PA-1 nd = not detected; Results: mean value ± standard deviation (*n* = 3); Different letters in the same row indicatesignificant differences (*p* < 0.05).

The tocopherol fraction of both seed oils was predominated by *β*-tocopherol (70.63%, and 76.61%, of the total tocopherols, PA-SB and PA-NB, respectively), and *γ*-tocopherol (28.44% and 23.42% of the total content, PA-SB and PA-NB, respectively). The peel oils showed completely different tocopherol profiles—*γ*-tocopherol was dominating in PA-SB

(69.74%), followed by *β*-tocopherol (30.32%), while *α*-tocopherol was detected as a single representative (100%) in PA-NB peel oil.

In the study of Ramadan [34], *β*- and *γ*-tocopherols were also the major tocopherols in the seed oil of *P. peruviana*, while *γ*- and *α*-tocopherols were the main components in the pulp/peel oil. No further parallel to other data could be made concerning the peel oil composition or the tocopherols, as to the best of our knowledge, there have been no other previous investigations in that direction.

#### *3.4. Determination of Amino Acids in Seed Cakes*

Accounting for the significant ratio of seeds in fresh fruit weight (Table 1) and their macronutrient indices (Table 2), the seed cakes resulting from oil extraction, otherwise considered a waste product, were considered a potentially valuable plant material worthy of recovery. Therefore, an attempt to provide new data in favor of their potential use and nutritional value was made in this study, by identifying some individual micro components in the seed cakes—amino acids and minerals. The results of the amino acid profile of seed cake proteins are presented in Table 5.

**Table 5.** Amino acid composition of seed cakes from *P. alkekengi* fruit phenotypes (PA-SB and PA-NB).

Results: mean value ± standard deviation (*n* = 3). Different letters in the same row indicate significant differences (*p* < 0.05). 1 essential amino acid.

As seen from the data, the dominant amino acids in the seed cakes of both the *P. alkekengi* phenotypes were arginine, aspartic acid, glycine, and alanine, with numerical deviations between the samples. Respectively, there were differences in the distributions of the rest of the amino acids, explicable by the varying conditions of the plant vegetation (phenotype). The ratio of essential amino acids (EAA) was relatively low and comparable in the two phenotypes, being about 0.4:1. Valine, phenylalanine, threonine, and isoleucine were present in slightly higher amounts than the other EAAs.

#### *3.5. Determination of Mineral Elements in Seed Cakes*

The results from the determination of 11 key macro and micro mineral elements in the seed cakes (Table 6) confirmed significant differences between the phenotypes only with regard to two of the macro minerals, sodium and calcium; the content of sodium was substantially higher in the PA-SB phenotype, and that of calcium was higher in the seed cakes of PA-NB fruit. The macrominerals potassium and magnesium were found in identical amounts in both phenotypes. The identified microminerals (Fe, Mn, Cu, Zn, Pb) showed comparable distributions between the phenotypes, with the single exception of Cr in PA-NB.

**Table 6.** Mineral composition of seed cakes from *P. alkekengi* fruit of two phenotypes (PA-SB and PA-NB).

Results: mean value ± standard deviation (*n* = 3). Different letters in the same row indicate significant differences (*p* < 0.05). 1 nd = not detected.

Evaluating the potential of the seed cakes for nutritional purposes, they could be considered a good source of potassium (supplying about 15% of the reference dietary intake (RDI) for men and about 12% for women; the RDIs being 2700 and 3500 mg, respectively), magnesium (about 60% of RDI for men and about 70% of RDI for women; 350 and 300 mg), as well as of iron (about 60% of RDI for men and about 30% of RDI for women; 8 and 18 mg), zinc (about 20% of RDI for men and about 30% of RDI for women; 11 and 8 mg), and other key microelements (copper and manganese) [40].

#### *3.6. Determination of Volatile Components in Fruit Concretes*

Another important aspect of the study was the objective of investigating the potential of *P. alkekengi* fruit for obtaining fruit concrete, a type of established aromatic product, widely used in perfumery and cosmetics [41]. Concretes are obtained by extracting fresh or dry plant materials with a non-polar solvent (e.g., hexane, petroleum ether, or benzene), followed by the complete removal of the solvent; the resulting concentrated aromatic product carries a specific fragrance profile, adding to the diversification of the fragrance nuances provided by the other types of aromatic products from the respective plant material, such as essential oils, absolute, resinoids, tinctures, and other extracts [41].

Accounting for the fact that the fruit pulp and peel are generally the vegetal material strongly related to fruit flavor, those parts of the *P. alkekengi* fruit were processed individually by *n*-hexane double extraction to obtain the respective fruit concretes. All concretes obtained in the study represented thick waxy masses with dark orange–yellow color.

The results revealed significant differences in the yield of fruit concretes, both between the phenotypes and between the fruit structural parts compared in the study. As seen from the data in Table 7, the yield of peel concrete was significantly higher than that of pulp concrete, thus clearly differentiating the potential of the two fruit structures with regard to their processing efficiency. In turn, there were significant differences in peel concrete yield on a phenotype basis; it was about three time higher in PA-NB phenotype, which was obviously related to the impact of environmental characteristics on plant metabolism.

The results from the identification by GC-MS analysis of individual aromatic volatiles in the obtained concretes and the total ion current (TIC) chromatograms with the major compounds (over 3%) are presented in Table 8 and Figure 3, respectively.

**Table 7.** Primary characteristics of fruit concretes obtained from two phenotypes of *P. alkekengi* (PA-SB and PA-NB).

Results: mean value ± standard deviation (*n* = 3). Different letters in the same row indicate significant differences (*p* < 0.05).

1 RT—retention time, min; 2 RI—retention index (Kovat's index); 3 TIC—total ion current; Results: mean value ± standard deviation (*n* = 3). Different letters in the same row indicate significant differences (*p* < 0.05). 4 nd = not detected.

Fifteen individual volatiles were identified by the applied GC-MS analysis in the pulp concrete of the PA-NB phenotype, accounting for 98.62% of the total content. The major components (in amounts over 3%) were: *β*-linalool (78.3%), *α*-pinene (6.9%), and *γ*-terpinene (5.5%). In the pulp concrete from the second phenotype, PA-SB, the number of identified compounds was 27 (98.56% of the total content), among which the major components were: *β*-linalool (61.9%), *γ*-terpinene (7.8%), α-pinene (5.3%), (5Z,9E)-farnesyl acetone (3.9%), neryl acetate (3.7%), and camphor (3.3%). The concrete obtained from the peel of the PA-NB phenotype contained 18 identified components (representing 98.41% of the total content). The major constituents in the product (over 3%) were: *β*-linalool (57.8%),

*α*-pinene (7.6%), *n*-tridecane (6.2%), sibirene (5.9%), camphor (5.5%), and *γ*-terpinene (5.5%). In turn, a total of 23 individual components were identified in the peel concrete from the PA-SB phenotype (98.72% of the content), with the major constituents being: *β*-linalool (49.6%), neryl acetate (11.4%), camphor (7.5%), germacrene D (6.9%), *α*-pinene (4.6%), and *tau*-cadinol (3.7%). *Molecules* **2022**, *27*, x FOR PEER REVIEW 13 of 17

**Figure 3.** *Cont*.

**Figure 3.** Chromatograms of volatiles in concretes obtained from two phenotypes of *P. alkekengi* fruit (PA-SB and PA-NB): (**a**) concrete from the peel of PA-SB phenotype; (**b**) concrete from the pulp of PA-SB phenotype; (**c**) concrete from the peel of PA-NB phenotype; (**d**) concrete from the pulp of PA-NB phenotype. **Figure 3.** Chromatograms of volatiles in concretes obtained from two phenotypes of *P. alkekengi* fruit (PA-SB and PA-NB): (**a**) concrete from the peel of PA-SB phenotype; (**b**) concrete from the pulp of PA-SB phenotype; (**c**) concrete from the peel of PA-NB phenotype; (**d**) concrete from the pulp of PA-NB phenotype.

The classification of the identified fruit concrete constituents revealed the presence of compounds belonging to different chemical classes (Figure 4). The distribution of the identified compounds (equaled to 100%) did not suggest considerable differences between the two structural elements of the fruit (pulp, peel), or between the phenotypes, although some specifics also existed. In all extracts, the volatile composition was dominated by oxygenated monoterpene derivatives (72.6% and 81.9% in the pulp and 71.4% and 68.4% in the peel for the PA-SB and PA-NB phenotypes, respectively), followed by monoterpene hydrocarbons (16.3% and 14.2% in fruit pulp; 8.4% and 15.5% in the peel). The comparison of the data obtained for the respective aromatic products from two phenotypes revealed significant differences in the contents of a number of minor and major components with known fragrance properties; for instance, components in the pulp concretes included nerol (1.7% in PA-SB and 0.1% in PA-NB), neryl acetate (3.7% in PA-SB, not detected in PA-NB), and (5Z,9E)-farnesyl acetone (3.9% in PA-SB, not detected in PA-NB); components in the peel concretes included neryl acetate (11.4% in PA-SB and only 1.1% in PA-NB), sibirene (5.9% in PA-NB, not detected in PA-SB), germacrene D (6.9% in PA-SB and only 0.2% in PA-NB), and *tau*-cadinol (3.7% in PA-SB, not detected in PA-NB).

Sesquiterpene hydrocarbons and oxygenated sesquiterpene derivatives were not detected in the concrete from the pulp of PA-NB phenotype, while diterpene representatives were found only in the concrete from the peel of PA-SB phenotype (3.0% of the identified content). The results revealed that the differences between the two phenotypes were more pronounced with regard to the extractible aromatic compounds in the peel compared with the fruit pulp. Thus, it could be presumed that fruit origin (phenotype) would not be a decisive factor for the organoleptic properties of the final product in *P. alkekengi* juice/pulp production, but would probably affect the usability of the resultant waste (peel or seed/peel residue); of course, additional and more targeted investigations are needed to support such assumptions, utilizing a wider range of sampling, parameter selection, and statistical tools. The classification of the identified fruit concrete constituents revealed the presence of compounds belonging to different chemical classes (Figure 4). The distribution of the identified compounds (equaled to 100%) did not suggest considerable differences between the two structural elements of the fruit (pulp, peel), or between the phenotypes, although some specifics also existed. In all extracts, the volatile composition was dominated by oxygenated monoterpene derivatives (72.6% and 81.9% in the pulp and 71.4% and 68.4% in the peel for the PA-SB and PA-NB phenotypes, respectively), followed by monoterpene hydrocarbons (16.3% and 14.2% in fruit pulp; 8.4% and 15.5% in the peel). Sesquiterpene hydrocarbons and oxygenated sesquiterpene derivatives were not detected in the concrete from the pulp of PA-NB phenotype, while diterpene representatives were found only in the concrete from the peel of PA-SB phenotype (3.0% of the identified content). The results revealed that the differences between the two phenotypes were more pronounced with regard to the extractible aromatic compounds in the peel compared with the fruit pulp. Thus, it could be presumed that fruit origin (phenotype) would not be a decisive factor for the organoleptic properties of the final product in *P. alkekengi* juice/pulp production, but would probably affect the usability of the resultant waste (peel or seed/peel residue); of course, additional and more targeted investigations are needed to support such assumptions, utilizing a wider range of sampling, parameter selection, and statistical tools.

**Figure 4.** Distribution of volatiles by chemical groups in concretes obtained from two phenotypes of *P. alkekengi* fruit (PA-SB and PA-NB). **Figure 4.** Distribution of volatiles by chemical groups in concretes obtained from two phenotypes of*P. alkekengi* fruit (PA-SB and PA-NB).

#### **4. Conclusions 4. Conclusions**

The results achieved by this study conducted on *P. alkekengi* fruit from Bulgaria add new details to the existing knowledge about the species. The study revealed that each of the fruit structural parts (peel, pulp, and seeds) or the resultant by-products (the seed cakes), rarely analyzed individually, had its specific features in terms of the assessed chemical characteristics, thus suggesting different options for their prospective use as sources of functional macro- and micronutrients—fiber, protein and some essential amino acids, oils and unsaturated fatty acids, tocopherols, and minerals. The study also provides new data on the obtaining and identification of the volatile profiles of *P. alkekengi* fruit concretes, thus contributing to the expansion of the range of available aromatic products. The presence of a number of aroma-active volatile compounds in the obtained peel and pulp concretes spoke in favor of a tangible potential for their future consideration as ingredients, e.g., in perfumery and cosmetic formulations. The results on the phenotyperelated differences presented in the study also suggested that fruit origin could, to a lesser or greater extent, affect the chemical composition of the assessed individual parts of *P. alkekengi* fruit and the aromatic products derived from them, especially if considering fruit properties on a wider geographical basis, in which genotype would most probably be another decisive factor. Based on these considerations, the outcomes from the study could be the grounds for future investigations in the indicated directions. The results achieved by this study conducted on *P. alkekengi* fruit from Bulgaria add new details to the existing knowledge about the species. The study revealed that each of the fruit structural parts (peel, pulp, and seeds) or the resultant by-products (the seed cakes), rarely analyzed individually, had its specific features in terms of the assessed chemical characteristics, thus suggesting different options for their prospective use as sources of functional macro- and micronutrients—fiber, protein and some essential amino acids, oils and unsaturated fatty acids, tocopherols, and minerals. The study also provides new data on the obtaining and identification of the volatile profiles of *P. alkekengi* fruit concretes, thus contributing to the expansion of the range of available aromatic products. The presence of a number of aroma-active volatile compounds in the obtained peel and pulp concretes spoke in favor of a tangible potential for their future consideration as ingredients, e.g., in perfumery and cosmetic formulations. The results on the phenotype-related differences presented in the study also suggested that fruit origin could, to a lesser or greater extent, affect the chemical composition of the assessed individual parts of *P. alkekengi* fruit and the aromatic products derived from them, especially if considering fruit properties on a wider geographical basis, in which genotype would most probably be another decisive factor. Based on these considerations, the outcomes from the study could be the grounds for future investigations in the indicated directions.

**Author Contributions:** Conceptualization, V.P., A.S., and S.E.; Formal analysis, V.P., Z.P., N.M., T.I., N.P., and M.S.; Funding acquisition, J.M.; Supervision, S.E., Z.O., S.S., and J.M.; Writing original draft, V.P., A.S., and S.E.; Writing—review & editing, Z.O., S.S., and J.M. All authors have read and agreed to the published version of the manuscript. **Author Contributions:** Conceptualization, V.P., A.S. and S.E.; Formal analysis, V.P., Z.P., N.M., T.I., N.P. and M.S.; Funding acquisition, J.M.; Supervision, S.E., Z.O., S.S. and J.M.; Writing—original draft, V.P., A.S. and S.E.; Writing—review & editing, Z.O., S.S. and J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support was provided by Tomas Bata University in Zlin, Faculty of Technology (IGA FT 2022/004). **Funding:** Financial support was provided by Tomas Bata University in Zlin, Faculty of Technology (IGA FT 2022/004).

**Institutional Review Board Statement:** Not applicable. **Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable. **Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the **Data Availability Statement:** The data presented in this study are available on request from the authors.

authors. **Acknowledgment:** The authors express their gratitude to Ivanka Dimitrova-Dyulgerova, Department of Botany, Plovdiv University "Paisii Hilendarski", Bulgaria, for the support in the **Acknowledgments:** The authors express their gratitude to Ivanka Dimitrova-Dyulgerova, Department of Botany, Plovdiv University "Paisii Hilendarski", Bulgaria, for the support in the collection and identification of the plant material.

collection and identification of the plant material. **Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**Appendix A**

**Figure A1.** Share of agricultural area in Carinthia situated in flood risk zones per municipality, 2018 (source of borders: Federal Office of Metrology and Surveying (BEV)).

**Figure A2.** Agricultural land use in flood-prone areas 2018, left side: Austria, right side: Carinthia.

**Figure A3.** Share of area for permanent settlement per municipality in Carinthia (own presentation; source of borders: Federal Office of Metrology and Surveying (BEV); source of area for permanent settlement: Statistics Austria).


**1. Introduction**

Decision making is a problem solving process that produces a goal of factors such as subjectivity and linguistics which tend to be presented in real life to a lower or greater level [1].  The results of these methods still contain uncertainty so that in this case fuzzy logic plays an important role in overcoming problems that contain uncertainty. Fuzzy logic is the basis of a system that can implement a problem and solve sharp

problems [2]. However, Fuzzy MADM is only able to solve the problem of uncertainty in the data presented and numbers of diverse attributes is usually conflicting, thus to make a decision there needs to be a classic MADM method, so that decisions are more precise and more accurate [3], besides this method can also be used to provide input to the doctor so that there is no mistake in diagnosing dengue disease. One of the classic MADM methods that can be used is Simple Additive Weighting.

Simple Additive Weighting is often referred as a method with weighted sum. The basic concept of SAW method is to find a weighted sum of performance branches on each alternative of all attributes [4]. One of the problems that can be solved by this method is the misdiagnosis of DHF. DHF is a type of infectious disease caused by the degue virus which is transmitted through the bite of the *Aedes aegypti* and *Aedes albopictus* mosquitoes. DHF is often misdiagnosed with Typoid Fever, Morbili, ARI, Ensafalitis and Acute Pharyngitis. These errors occur because the initial symptoms that arise from the five diseases are almost the same as DHF [5]. However, in this case the application of SAW method is less effective if a Decision Support System is made so that a development method is needed. The development method that can be used is the FMADM method with its development or often called Fuzzy Decision Making (FDM). This method is development method of the classic MADM method. The results of SAW method will be used as a level of importance or input on the FDM method. The combination of these two methods will produce more optimal output.

### **2. Methodology and realization**

#### **2.1 Designing FMADM with SAW and FDM**

The data used are primary and secondary data, primary data obtained from the results of doctor interviews and secondary data is data on patients with DHF, secondary data will be used to validate the system. Completion of cases of dengue diagnosis will be through SAW method then the results of SAW method are used in the FDM method.

The first method will use one crisp value with 1 degree membership and use preference weight multiplication while the second method uses 3 crisp values namely right boundary, left boundary and crisp value with 1 membership degree which will later go through the aggregation process and total integral value.

#### **2.2 The FMADM method with SAW to diagnose a type of disease**

Completion using the FMADM method with SAW:

#### *2.2.1 Determine alternative sets and criteria*

Alternative (Ai) is a1 = Morbili, a2 = DBD, a3 = ARI, a4 = Typoid fever, a5 = Acute pharyngitis, a6 = Ensafalitis. Ci criteria are c1 = Fever, c2 = Spots, c3 = Bleeding gum, c4 = Nausea, c5 = Headache, c6 = Defecation Disorders

#### *2.2.2 Determine the criteria weight*

The weight of the criteria is obtained from triangular fuzzy numbers which are then converted into the form of crisp.

*Fuzzy Multi-Attribute Decision Making (FMADM) Application on Decision Support… DOI: http://dx.doi.org/10.5772/intechopen.94614*

#### *<IP_ADDRESS> Fever*

The author defines the universal value for the criteria for fever is [0,1] and divides it into 5 categories of fuzzy triangle sets, which are normal (N), low fever (DR), moderate fever (DS), high fever (DT), very high fever (DST).

By using the concept of the Likert scale and the defuzzy method, Large of Maximum, **Table 1** is obtained as the weight of the criteria for fever.

### *<IP_ADDRESS> Spots (Petheciae)*

The author defines the universal value for the criteria of spots is [0,1] and divides them into 5 categories of fuzzy triangle sets which are none (TA), few (SDK), somewhat a lot (ABYK) many (BYK), very much (SBYK). By using the concept of the Likert scale and the defuzzy method, Large of Maximum, the **Table 2** is obtained as the weight of the criteria for spots:

#### *<IP_ADDRESS> Bleeding gum*

We are defines the universal value for bleeding gum criteria is [0,1] and divides it into 2 fuzzy triangle set categories namely never (TP), ever (P). By using the concept of the Likert scale and the defuzzy method, Large Of Maximum, the **Table 3** is obtained as the weight of the bleeding gum criteria.

#### *<IP_ADDRESS> Nausea*

The author defines the universe value for the nausea criteria is [0.1] and divides it into 4 fuzzy triangle set categories namely never (TP), ever (P), rare (J) and often (S). By using the concept of the Likert scale and the defuzzy method, Large of Maximum, **Table 4** is obtained as the weight of the criteria for nausea.

### **Table 1.**

*Weight of fever.*

**Table 2.** *Weigth of spots.*

**Table 3.**

*Weight of bleeding gum.*

**Table 4.** *Weigth of nausea.*


3.2.2. Cell Motility Assay for Wound Healing

Microscopy images of gap closure results are reported in Figures 5–7. ALI and GR samples were tested at 50% (*v*/*v*) concentration, while PS9, G30, PS9ALI, PS9GR, G30ALI and G30GR were tested at 50 μg/mL of clay mineral. The microphotographs taken at zero time (0 h) showed for all the samples the presence of defined gaps mimicking wounds. Normal fibroblasts grown at confluence were clearly visible in each side of the wound. The time 0 gaps were reported to measure approximately 500 μm, which was in agreement with the variability of the silicone inserts of the Petri μ-Dishes used (500 ± 50 μm). Insoluble clay particles were visible in the cultures treated with PS9, G30 and hydrogels PS9ALI, PS9GR, G30ALI and G30GR, unlike GM, ALI and GR. In all cases, after 24 h, fibroblasts started to invade the gap and even established contact with the cells of the opposite side

in certain points of some samples. NHDF cells also maintained their typical fusiform morphology during the whole experiment. These facts confirmed that the presence of spring waters, clay or hydrogels did not impair cell growth. This was in line with the biocompatibility results previously discussed. In all cases, fibroblasts crossed the empty zone after 48 h, thus forming anastomosis. In spring water ALI and GR samples, fibroblasts of both sides of the wound established contact within 24 h (Figure 5). In comparison with the control (GM), it was clear that the addition of ALI and GR favored wound closure, since the contact points between fibroblasts were more numerous. According to the measurements of the wounded area performed by image analysis, ALI and GR samples covered, respectively, 62.9% and 59.1% of the initial wounded area, whereas GM covered about 31.8% of the wound (Figure 8). After 48 h all the cell substrates reached confluence, and this further supports the biocompatibility of the hydrogels and their components in vitro. Previous works have claimed that the presence of elements such as B3+ and Mn+2 in keratinocyte culture mediums induced the migration of these cells [30]. They tested boron and manganese at different concentrations (500–1000 μg/L and 100–1500 μg/L, respectively). In fact, keratinocytes in contact with these elements were able to cover the artificial gap (scratch-assay method) with 20% more efficiency with respect to the control group (without B3+ and Mn+2) in 24 h. ALI and GR chemical compositions have been previously reported by Aguzzi et al. [111] and García-Villén et al. [63] (Table 3). The absence of significant differences between GR, ALI and GM groups further confirmed the biocompatibility of both mineral medicinal waters since they did not hinder wound closure. The presence of B3+ and Mn+2 in both GR and ALI could explain why they did not interfere with cell motility during wound healing in a significant manner. Other elements such as As3+ and Fe2+ (both presents in ALI and GR waters) have also demonstrated to improve wound healing, particularly of the nasal mucosa [112]. General skin regenerative properties of spring waters have been reported in the literature [32–34,113]. The study of Liang et al. demonstrated that skin regenerative properties of Nagano spring water were related to the spring water chemical composition, with no influence of microorganisms [31]. Another beneficial effect that could be ascribed to ALI and GR according to their chemical composition is the protective effect against oxidative stress due to the presence of sulfur [114].

**Figure 5.** Optical microscopy of wound healing tests for control group (GM) and spring waters (ALI and GR) at 50% (*v*/*v*) culture concentration.

**Figure 6.** Optical microscopy of wound healing tests for control group (GM), PS9 clay mineral and corresponding peloids PS9ALI and PS9GR, all of them with a clay concentration of 50 μg/mL.

**Figure 7.** Optical microscopy of wound healing tests for control group (GM), G30 clay mineral and corresponding peloids G30ALI and G30GR, all of them with a clay concentration of 50 μg/mL.

**Figure 8.** Histogram of % wound reduction after 24 h, calculated according to Equation (2). Mean values ± s.d. (n = 6). Significant differences between samples and GM are marked with \*.

Regarding PS9 and G30 clay samples (Figures 6 and 7), it was not possible to find zones with full fibroblast confluence after 48 h, though both sides of the wound were able to establish contact at this time. Fibroblast contacts of PS9 and G30 samples in the culture medium were slower with respect to GM. According to MTT results (Section 3.2.1), which were also performed for 24 h, pristine clays did not hinder cell viability. However, their presence seemed to impede fibroblast mobility during the wound healing assay, slowing down the total gap closure with respect to GM and studied peloids. In fact, the uncovered gap width after 24 h was significantly higher for PS9 and G30 with respect to the rest of the samples (Figure 8), thus indicating a slower coverage of the wounded area. For these similar samples, the majority of insoluble particles visible within the cell substrate were concentrated adjacent to or over/inside fibroblasts. It is well known that cell membranes are negatively charged and can be penetrated by positive substances. Negatively charged particles can also interact and even penetrate cells by endocytosis-mediated mechanisms [102,103,115]. According to the zeta potential results previously reported (Figure 2), PS9 and G30 had a negative net charge in the major part of the pH range tested. These clay particles could interact between them (thus forming bigger aggregates) and with cells by establishing interactions such as Van der Waals forces. This hypothesis was the starting point of Abduljauwad and Ahmed, who proposed that the interaction between clay minerals and cells would hinder cell migration [116]. They evaluated the roles of montmorillonite, hectorite and palygorskite particles in cancer cell migration and demonstrated these materials could prevent cellular metastasis. Results of scratch-induced wound healing assays reported by these authors demonstrated that clay mineral particles significantly delayed the gap closure in comparison with control experiments. For instance, while the control showed full gap closure within 24 h, palygorskite showed a mean gap closure of 59 ± 3% after 24 h. Polymeric composite films containing montmorillonite were evaluated by Salcedo et al. [50] and Mishra et al. [117]. The scratch assay showed, in both cases, that the presence of montmorillonite particles alone was able to alter cell behavior (Caco-2 and fibroblasts, respectively for each study), slowing down the gap closure. Similar results were reported by Vaiana et al. on montmorillonite and keratinocytes [118]. These results were in agreement with PS9 and G30 performances during wound healing: though clays were not cytotoxic, more time would be necessary to obtain complete cell confluence within the artificial wound (gap) in their presence. The biocompatibility and safety of natural clay minerals such as palygorskite during wound healing was also supported by in vivo studies. A natural Brazilian palygorskite was tested for in vivo wound healing of rats [119]. In comparison with functionalized clay minerals, the natural one provided more advanced and safer wound healing, since histological cuts demonstrated the presence of dermal papilla and hair follicles after 14 d of treatment [119].

The presence of hydrogels promoted in vitro fibroblast mobility during wound healing processes. In fact, coverage of the artificial gap was faster for PS9ALI, PS9GR, G30ALI and G30GR with respect to GM and pristine clays (Figure 8). It is worth to note that all therapeutic muds were used in concentrations equal to the powdered clay samples. That is, the amount of peloid within the culture medium was higher in order to compensate for them only possessing a 10% *w*/*w* of clay mineral. Whatsoever the nature of the interaction between clay particles and fibroblasts, responsible for the gap closure deceleration (observed in PS9 and G30 tests), it was reduced to a minimum when it came to thermal mud formulations. In fact, significant differences were found between gap closures of peloids vs. GM, demonstrating that the evaluated hydrogels induced a positive effect during wound healing.

These results are supported by previous studies in which clay minerals have reported to exert neutral or positive wound healing effects when combined with other substances [44,120]. For instance, no significant changes have been found during in vitro wound healing studies of montmorillonite–chitosan–silver sulfadiazine nanocomposites with respect to the control. That is, the presence of the clay mineral did not hinder the gap closure procedure and did not affect fibroblast phenotypes [54]. On the other hand, better skin re-epithelialization and reorganization have been found when halloysite and chitosan were combined to form a nanocomposite with respect to the use of both materials independently [47]. In vivo infected wound treatment with silver nanoparticles was improved by the use of montmorillonite as a carrier, which reduced the drug cytotoxicity [121]. Montmorillonite also demonstrated to exert a wound healing effect when combined with chitosan and polyvinylpyrrolidone polymers [48]. The nanocomposite films containing bentonite increased the in vivo wound healing processes in mice when compared with the formulations without the clay. For instance, wound closure after 16 d was 92–93% for samples without clay and 95–97% for those with montomorillonite, and all of them were higher than the negative control at the same time (84%) [48].

Despite the scarce literature regarding the use of therapeutic muds in skin regenerative properties, the existing studies were also in agreement with the present observations. Thermal mud treatments with volcanic deposits of Azerbaijan facilitated the healing of chronic gangrenous wounds of diabetic patients [60]. The major part of the patients subjected to pelotherapy reached total wound recovery by the end of the treatment. Two natural Dead Sea black mud samples were evaluated by Abu-al-Basal for their in vivo wound healing properties [61]. One of them was used in its pristine state, while the other was formulated in form of a facial mask by adding plant extracts and vitamin E. Both samples accelerated the wound healing process by enhancing granulation, wound contraction, epithelialization, angiogenesis and collagen deposition. Another in vivo wound healing study was performed by Dário et al. [62]. They evaluated a natural peloid extracted from Ocara Lake (northeast of Brazil), which was sieved, solid-state characterized and sterilized. The solid fraction was mainly composed of quartz, illite and kaolinite, and it was formulated as an emulsion. The emulsion was put in contact with injured Wistar rats. The formulation including the Ocara Lake solid fraction induced histological changes in the deep dermis that allowed an effective healing with respect to control groups. On the other hand, there is evidence in literature about the anti-inflammatory properties of peloids [122]. Moreover, a recent study focusing on fibrous clays suggests that sepiolite and palygorskite, the main components of PS9 and G30, respectively, did not influence the in vitro NO (inflammation signal) production of murine macrophages (RAW 264.7). Furthermore, both clays decreased the leucocyte infiltration shortly after exposure in vivo on mouse ear edema (12-O-tetradecanoylphorbol-13-acetate (TPA) as inflammatory agent). In particular, sepiolite and palygorskite caused decreases on the number of infiltrated cells per field [59]. Analogously, other clay minerals, as halloysite and montmorillonite, proved macrophage cytocompatibility and negligible secretion of TNFa, as proinflammatory cytokines [123]. The anti-inflammatory properties of clays could control the inflammatory phase in the healing process, shortening healing time and avoiding wound chronicity.

#### *3.3. Confocal Laser Scanning Microscopy*

CLSM microphotograph results (Figure 9) showed fluorescence of fibroblast nuclei and cytoplasm. Green fluorescence is due to the binding of phalloidin specifically with F-actin, and the blue one is due to 4',6-diamidino-2-phenylindole (DAPI) bounded to DNA. Morphology of fibroblasts during wound healing is very important, since it gives information about migration and adhesion of cells. F-actin filaments facilitate cell–cell interaction and tissue regeneration [124]. In all samples, regardless of the type of sample with which fibroblasts were put in contact with, NHDF showed typical fusiform morphology. Moreover, fibroblasts located in the wounded area possessed typical morphology of migrating cells. Therefore, it was possible to identify retraction/protrusion parts of fibroblasts due to the position of F-actin. Typical actin filament structures were detectable in all samples: lamellipodia, filopodia and stress fibers (dorsal stress fibers and transverse arcs). Moreover, fibroblasts in mitosis were also visible in some points.

Regarding the speed of wound closure, the CLSM results were in agreement with those obtained by optical microscopy. GM control after 24 h demonstrated that NHDFs were able to make contact and started to close the gap, though yet some empty zones were clearly visible. The number of cells inside the wound gap in ALI and GR samples was higher than that in GM. PS9 and G30 showed the worst result in terms of "gap closure", which confirmed the previous results. Once again, it was possible to observe that the "slowing down" effect of PS9 and G30 did not happen when both clays were formulated in form of nanoclay/spring water hydrogels. Particularly, hydrogels G30ALI, G30GR, PS9ALI and PS9GR induced a faster wound healing with respect to the rest of the samples.

**Figure 9.** *Cont.*

**Figure 9.** CLSM microphotographs during wound healing. NHDMs were stained with phalloidin-FITC (green, F-actin filaments) and DAPI (blue, nucleus). Green arrows indicate the migration direction; different F-actin structures have been identified by L (lamellipodia), F (filopodia), SF (stress fibers) and DSF (dorsal stress fibers). Additionally, M stands for (mitosis) and CP stands for (clay particles).