Antioxidative and Photoprotective Activity of Pinus nigra, Pinus strobus and Pinus mugo

Abstract

Substances that delay the skin aging process have become very popular lately. Undoubtedly, this is influenced by all kinds of efforts to maintain a youthful appearance for as long as possible. Plant-derived antioxidants are a group of compounds that exhibit protective properties against the degenerative effects of oxidative stress on skin aging. Another important factor that protects skin against aging is photoprotective agents. The comparison of antioxidant and photoprotective activities seems to be interesting. The aim of this study was to evaluate the antioxidant properties of Pinus strobus, Pinus nigra, and Pinus mugo extracts using two frequently applied methods, i.e., DPPH and ABTS. Moreover, the polyphenol content was evaluated using Folin–Ciocalteu method. The correlation between the polyphenol content, antioxidant potential of the extracts, and sun protection factor in vitro was evaluated. Extracts were prepared using methanol, ethanol, isopropanol, and n-propanol in three concentrations: 40% (v/v), 70% (v/v), and undiluted. Ultrasound-assisted extraction, which is a type of green extraction technique, was applied for 15, 30, or 60 min. The highest antioxidant activity determined by the DPPH and ABTS methods was observed for Pinus mugo extracts in 40% ethanol and 40% methanol, respectively, both after 15 min extraction. The highest total polyphenol content was also found in Pinus mugo extracts. These activities were significantly higher than those of Pinus strobus and Pinus nigra. Similarly, the highest SPF values were also found for Pinus mugo extracts. Moreover, a strong correlation was observed between the antioxidant potential and SPF—the highest values were found for the correlation between the SPF and antioxidant activity determined using the ABTS method. Based on the obtained results, Pinus mugo could be suggested as a possible component for use in cosmetics.

1. Introduction

The human body, the outermost part of which is the skin, is constantly exposed to many harmful external factors, including solar radiation, which can lead to premature aging. Skin aging is an irreversible and inevitable physiological process that is influenced by many factors including mechanical damage due to facial expression; biological factors, mainly caused by genetically determined hormonal changes; environmental factors, such as active or passive smoking and air pollution; and factors connected with lifestyle, which include, among others, diet, skincare, stress, and disease [1,2]. Another very important factor that accelerates skin aging is UV radiation, which is responsible for as much as 80% of its occurrence [3]. Excessive exposure to ultraviolet radiation is associated with serious health risks such as UV-induced skin damage, skin photoaging (atrophy, pigmentary changes, and wrinkles), solar sunburn, skin sensitization, and malignancy [4].

Another important factor which should be taken into account is population aging. In most countries, the number of older people is increasing rapidly. Moreover, this trend seems to be accelerating [5]. For instance, according to the results of the Seventh National Census in China, the population over 60 years has reached 264 million [6]. It is expected that, between 2015 and 2050, the proportion of the world’s population over 60 years will nearly double from 12% to 22% [6]. As age is an indisputable major risk factor of skin cancer and the number of skin cancer cases is expected to increase, early prevention seems to be of importance [7]. For the older population, the application of sunscreens as well as antioxidants is important.

UV radiation can initiate the formation of free radicals, which may be responsible for the damage of many skin cells and components, such as fibroblasts, keratinocytes, collagen, and elastin [8]. Antioxidants are a group of compounds which can interrupt the formation of free radicals and protect people from the above-mentioned diseases [3]. Many people pay attention to their appearance to ensure better well-being and better acceptance by others; they are looking for products containing antioxidants, i.e., ingredients that have beneficial effects on appearance. Substances that delay the skin aging process have become very popular lately. Undoubtedly, this is influenced by all kinds of efforts to maintain a youthful appearance for as long as possible [4]. People attach great importance to improving their image and, to this end, are looking for products containing antioxidants, which are ingredients that provide beneficial effects. Moreover, plant-derived antioxidants are a group of compounds that exhibit protective properties against the degenerative effects of oxidative stress on skin aging [9,10]. Their beneficial effects on the appearance and function of the skin have led to a search in recent years for new substances with antioxidant potential among plant raw materials [9]. There are many plants widely used for this purpose. Many trees, including conifers, contain components that exhibit antioxidant properties [11,12]. Such trees include, among others, those of the Pinaceae family: Pinus strobus, Pinus nigra, and Pinus mugo [13,14].

Pinus strobus is a species of coniferous tree in the Pinaceae family, which mainly grows in the northern and eastern areas of North America (Canada and the USA) [15] and in the Carpathian Mountains in the Czech Republic and southern Poland [16]. Pinus strobus is a tall tree that can reach up to 50 m in height. The needles of the Pinus strobus are long, gathered in bundles of five needles [17], have an intense green color, and can reach up to 15 cm in length. They are flexible and soft compared to the needles of Pinus nigra and Pinus mugo. Extracts of Pinus strobus needles are rich in active ingredients such as terpenes, flavonoids, phenolic acids, vitamins, and other bioactive compounds that have beneficial properties and can be used in medicine and cosmetology. The main component of the needles is alpha-pinene [18]. In medicine, like other pine species, it is used to treat the respiratory tract, has an expectorant and antiseptic effect, and supports the immune system. In cosmetology, it is used to slow down aging and to moisturize the skin. Like the other trees from the Pinaceae family, it is used in aromatherapy due to its relaxing effects [19].

Pinus nigra is an evergreen and long-lived tree of the Pinaceae family, which is found in the southern Balkans and Turkey, usually reaching 30 m to 40 m in height. Needles grow in pairs, are 8–16 cm long and 1–2 mm in diameter and can be straight or curved. They usually stay on the tree for 3–4 years but sometimes last for up to 8 years [20,21]. Pine wood is a very valuable natural raw material. Although data on Pinus nigra are scarce, its antibacterial, antioxidant, and antiproliferative activity has been found [22] It can be used in medicine and cosmetology [23]. Essential oil extracted from Pinus nigra contains mainly α-pinene (about 50%). This oil has anti-inflammatory, antiseptic, and relaxing effects on physical and mental fatigue. In aromatherapy, it has a calefacient effect especially for rheumatic pains [19,24]. Pine extracts are rich in terpenes, polyphenols, catechins, epicatechins, and taxifolin, and have good antioxidant properties and support the cardiovascular and respiratory systems. Pinus nigra needles contain large amounts of polyphenols and flavonoids. It was found that the antioxidant capacity of these needles is higher compared to other species of evergreen trees in the Pinaceae family [22,25].

Pinus mugo is a species of shrub or tree, whose main area of occurrence is the mountain regions of the Central and Southern Europe. Mountain pine grows at altitudes from 200 m to 2700 m. Pinus mugo is a low, coniferous tree with many stems. It reaches 3–6 m in height. P. mugo needles have many beneficial properties. They have wax on their surface, which allows the accumulation of lipophilic organic compounds [26]. Extracts from needles, bark, buds, and cones, as well as essential oils, contain large amounts of secondary metabolites, which can be applied in respiratory and rheumatic diseases, as well as anti-inflammatory and expectorant agents. These extracts are rich in active ingredients, including volatile compounds, higher alcohols, vitamins, minerals, and tannins. In addition, due to the polyphenols content, P. mugo extracts have high antioxidant activity, which gives them anticancer and antiproliferative properties [27,28].

Another important factor for protecting an organism against aging is the use of photoprotective agents [29,30]. Most of the preparations available on the market are based on synthetic compounds. However, there is little information regarding the widespread use of plant extracts. As mentioned, such extracts also have antioxidant properties, which would be beneficial for human health [31].

Due to the variety of raw materials containing substances with valuable health-promoting properties, a number of extraction methods can be used to isolate active ingredients. The selection of an appropriate method should ensure a fast process and the isolation of compounds of the highest possible activity, should avoid degradation or change in the properties of the isolated valuable ingredients, and, if possible, should minimize the co-extraction of undesirable compounds, especially contaminants. For this purpose, classic extraction techniques, such as maceration, digestion, percolation, or Soxhlet extraction, are often used [32,33]. In order to eliminate certain unfavorable features of these methods, modern extraction methods, classified as so-called green extraction techniques, which are eco-friendly and economical, are increasingly used. These methods include supercritical fluid extraction, pressurized liquid extraction, ultrasound-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction, and others [33,34]. One of these green techniques is ultrasound-assisted extraction, used in the current study [35]. This technique is based on the application of ultrasonic waves to a matrix immersed on a liquid medium, producing the rupture of cell walls and releasing the compounds of interest [36]. According to Dzan et al. this method does not only contribute to the increased extraction yield of polyphenols, but also better preserves and increases the biological activity of polyphenol extracts compared to traditional maceration and Soxhlet extraction [37].

In the present study, we have decided to evaluate the antioxidant properties of the alcoholic extracts of the three species of the Pinaceae family, i.e., Pinus strobus, Pinus nigra, and Pinus mugo. Antioxidant activity was determined using the DPPH and ABTS methods, whereas the polyphenol content was evaluated using the Folin–Ciocalteu technique. There is a lack of reports on the photoprotective parameters of these three trees. Therefore, the antioxidant potential of these extracts has been compared with their selected sun protection parameters determined in vitro.

2. Materials and Methods

Reagents. DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ehylbenzothiazline-6-sulphonic acid) diammonium salt were purchased from Sigma-Aldrich (St. Louis, MO, USA); Folin–Ciocalteu reagent was purchased from Merck, Darmstadt, Germany). Methanol, ethanol, propan-1-ol, propan-2-ol, sodium persulphate, and sodium carbonate anhydrous were from Chempur, Piekary Śląskie (Poland). Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) and gallic acid were from Sigma-Aldrich. All the reagents were of analytical grade.

Raw material. The plant material applied in this study was collected from a natural state in July 2024. It consisted of needles of Pinus strobus (53°58′58″ N, 15°56′46″ E), Pinus mugo (54°00′28″ N, 15°59′50″ E), and Pinus nigra (54°00′19″ N, 15°58′54″ E), growing near Białogard (West Pomeranian Voivodeship, Poland). All raw material was dried at room temperature in a dark, ventilated place. Samples of raw material marked as PS-01/2024 PM-01/2024, and PN-01/2024, respectively, were deposited in Department of Cosmetic and Pharmaceutical Chemistry, Pomeranian Medical University in Szczecin.

Extraction of plant material

Extracts were prepared in four short-chain alcohols, i.e., methanol, ethanol, propan-1-ol, and propan-2-ol, as previously described [38,39]. All alcohols were applied in three concentrations: 40% (v/v), 70% (v/v), and undiluted. The extracts were prepared as follows: to 0.5 g of ground raw material in glass-stoppered tubes, 10 mL of the above-mentioned solvent was added. All the tubes were subjected to ultrasound-assisted extraction (Sonic 0.5, Polsonic, Warsaw, Poland) working at a frequency of 40 kHz for 15, 30, or 60 min. Thereafter, the remaining plant material was separated by filtration and clear extract was transferred to tightly closed plastic tubes and stored in a dark place at room temperature until further analysis, which include evaluation of antioxidant activity and photoprotection parameters.

Evaluation of antioxidant activity of the extracts

To determine antioxidant activity of the extracts, two frequently applied methods based on DPPH and ABTS radical reduction were used, as described in our previous papers [2,40,41]. Shortly, determination of antioxidant activity using DPPH method include preparation of DPPH solution in 96% (v/v) ethanol. For this purpose, 0.012 g of DPPH was dissolved in 100 mL of ethanol using magnetic stirrer. To obtain working solution of absorbance, 1.00 ± 0.02 at 517 nm in 1 cm cuvettes, stock solution was diluted with 70% (v/v) ethanol. To 2500 µL of working solution of DPPH, 132 µL of the sample was added and thoroughly mixed. After 10 min incubation in dark at room temperature, absorbance at 517 nm was measured using Hitachi U-5100 spectrophotometer (Tokyo, Japan) [38,40]. Each extract was evaluated in 3 replicates. The antioxidant activity of the samples was expressed as trolox equivalent antioxidant capacity (TEAC) in mg trolox/g raw material [41]. For this purpose, calibration curve of absorbance vs. trolox concentration was prepared (y = −1.1018x + 0.9712, R² = 0.9926).

To evaluate antioxidant activity using ABTS method, to 2500 µL of 7 mM ABTS solution of absorbance 1.00 ± 0.02 at 734 nm in 1 cm cuvettes, an aliquot of 25 µL of the extract was added and thoroughly mixed. The samples were then incubated at room temperature for 6 min. Absorbance was measured at 734 nm. Three samples were prepared for each extract [2,42]. The antioxidant activity of the samples was expressed as trolox equivalent antioxidant capacity in mg trolox/g raw material [41]. For this purpose, calibration curve of absorbance vs. trolox concentration was prepared (y = −0.2395x + 0.9783, R² = 0.9936). The results are expressed as mean ± standard deviation (SD).

The polyphenol content in examined extracts was evaluated using Folin–Ciocalteu method [43] as described in our previous papers [42,44]. In short, to 150 µL of examined extract, 150 µL of tenfold diluted commercial Folin–Ciocalteu reagent (Merck) was added followed by adding 1350 µL of sodium carbonate aqueous solution and the same volume of water. The tubes were thoroughly mixed and incubated for 15 min at room temperature. After this time, the spectrophotometric measurement at 765 nm was performed. The polyphenol content in the samples was expressed as gallic acid equivalents in mg gallic acid/g raw material [42,44]. For this purpose, calibration curve of absorbance vs. gallic acid concentration was prepared (y = 0.0175x + 0.0102, R² = 0.9902). The results are expressed as GAE (gallic acid equivalent) in mg gallic acid/g raw material.

The in vitro SPF (sun protection factor) was evaluated according to the method previously described by others [45,46,47]. Absorbance spectrum was taken in 1 cm quartz cuvettes using studied extracts diluted as follows: to 75 µL of extract, 2000 µL of solvent used for extraction was added. UV spectra were taken from 290 nm to 320 nm using Hitachi U-5100 (Tokyo, Japan) and DR6000 Hach (Hach-Lange, Wrocław, Poland) spectrophotometers. SPF was calculated using Masur equation:

$$SPF=CF\sum _{290}^{320}EE(\mathit{\lambda })·I(\mathit{\lambda })·A(\mathit{\lambda })$$

(1)

where CF is the correction factor (=10), EE(λ) is the erythmogenic effect of radiation with wavelength λ, and A(λ) is the absorbance at wavelength λ. The absorbance was determined at 290–320 nm every 5 nm. The values EE(λ) · I(λ) are constants, and were determined by Sayre et al. [48]. Table 1 presents these values also used in this study to evaluate SPF value of studied extracts.

Table 1. Values of EE(λ) · I(λ) [45,46].

Wavelength [nm]	EE(λ) · I(λ)
290	0.0150
295	0.0817
300	0.2874
305	0.3278
310	0.1864
315	0.0839
320	0.0180

Table 1. Values of EE(λ) · I(λ) [45,46].

Wavelength [nm]	EE(λ) · I(λ)
290	0.0150
295	0.0817
300	0.2874
305	0.3278
310	0.1864
315	0.0839
320	0.0180

The UVA/UVB ratio was calculated using calculated areas under absorption spectrum in UVA region, i.e., 320–400 nm, and UVB region, i.e., 290–320 nm. The following equation was applied:

$$\mathit{UVA}\mathit{/}\mathit{UVB}\mathit{=}\mathit{\left(}{ʃ}_{320}^{400}A\right(\mathit{\lambda })d\mathit{\lambda }\mathit{/}{ʃ}_{320}^{400}d\mathit{\lambda }\mathit{)}\mathit{/}\mathit{\left(}{ʃ}_{290}^{320}A\right(\mathit{\lambda })d\mathit{\lambda }\mathit{/}{ʃ}_{290}^{320}d\mathit{\lambda }\mathit{)}$$

(2)

These values could be applied to describe protection category according to the Boots Star Rating System [45]. Value of 0–0.2 is described as no protection, 0.21–0.4 as minimal, 0.41–0.6 as moderate, 0.61–0.8 as good, 0.81–0.9 as superior, and values above 0.91 as ultra.

The critical wavelength λ_cr was calculated according to the following equation:

$$\underset{290}{\overset{\lambda cr}{ʃ}} A(\lambda )·d\lambda =0.9·\underset{290}{\overset{400}{ʃ}} A(\lambda )·d\lambda$$

(3)

where λ_cr is critical wavelength. According to [47] both parameters, i.e., UVA/UVB ratio and the critical wavelength λ_cr, are very reproducible even if different substrates are applied.

Statistical analysis.

The results are presented as mean ± standard deviation (SD). As some groups of results differs from the normal distribution (checked by Lilliefors test), to evaluate the differences between the groups of results, non-parametric tests, i.e., Kruskal–Wallis and Mann–Whitney tests, were applied. Moreover, linear regression between the groups was also evaluated and correlation coefficients and their statistical significance were calculated. All the calculations and figures were carried out using ProStat v. 5.5 and Excel programs.

3. Results

Figure 1 presents the antioxidant activity of Pinus strobus alcoholic extracts evaluated using the DPPH method. The higher activity was observed for extracts in diluted alcohols—they were comparable in the same solvents regardless of the extraction time. The highest activity of 3.85 ± 0.02 mg trolox/g raw material (TEAC) was found for 70% ethanol used as an extractant after 30 min of extraction and for 70% methanol after the same extraction time (3.81 ± 0.07 TEAC). The lower activity was found for extracts in undiluted alcohols; the lowest activity was observed for extracts in undiluted n-propanol (1.24 ± 0.10 TEAC) and undiluted isopropanol (1.26 ± 0.10 TEAC), both after 30 min of extraction.

Figure 2 presents the antioxidant activity of Pinus mugo alcoholic extracts evaluated with the DPPH method. Similarly to Pinus strobus extracts, the higher activity was observed for extracts in diluted alcohols—they were comparable in the same solvents regardless of the extraction time. The highest activity of 14.52 ± 0.26 TEAC was found for extracts in 40% ethanol after 15 min of extraction and in 40% methanol after 30 min of extraction (14.49 ± 0.46 TEAC). The lower activity was found for extracts in undiluted alcohols; the lowest activity was observed in extracts in undiluted n-propanol (2.13 ± 0.09 TEAC) after 60 min of extraction and in undiluted isopropanol (2.37 ± 0.32 TEAC) both after 30 min of extraction. Statistically significant differences were found between the antioxidant activity evaluated with the DPPH method between Pinus strobus and Pinus mugo extracts (p < 0.001).

The antioxidant activity of Pinus nigra alcoholic extracts determined by using the DPPH method is presented in Figure 3. Similarly to Pinus strobe and Pinus mugo extracts, the higher activity was found for extracts in diluted alcohols—they were comparable in the same solvents regardless of the extraction time. However, higher activity was observed in the extract obtained after one hour of extraction. The highest antioxidant potential of 3.74 ± 0.01 TEAC was found for 40% methanol used as a solvent after 60 min of extraction and for 40% n-propanol and ethanol after 60 min of extraction—3.62 ± 0.03 and 3.59 ± 0.13 TEAC, respectively. The lower activity was found for extracts in undiluted alcohols; the lowest one was observed for extracts in undiluted isopropanol (0.43 ± 0.16 TEAC) after 15 min of extraction and in undiluted n-propanol (0.55 ± 0.08 TEAC) after 30 min of extraction. Statistically significant differences were found between the antioxidant activities evaluated with the DPPH method between Pinus nigra and Pinus mugo extracts (p < 0.001). as well as between Pinus nigra and Pinus strobus extracts (p < 0.001).

Figure 4 presents the antioxidant activity of Pinus strobus alcoholic extracts evaluated using the ABTS method. The higher activity was observed for extracts in diluted alcohols—they were comparable in the same solvents regardless of the extraction time. The antioxidant activity of extracts in undiluted alcohols increase in some cases with the extension of extraction time. The highest activity of 19.34 ± 0.10 TEAC was found for 40% ethanol used as a solvent after 15 min of extraction and for 70% methanol after the same extraction time (17.56 ± 0.62 TEAC). The lower activity was found for extracts in undiluted alcohols; the lowest was observed for extracts in undiluted isopropanol (1.41 ± 0.28 TEAC) and in undiluted n-propanol (2.99 ± 1.12 TEAC), both after 15 min of extraction. The antioxidant activities evaluated with the ABTS method were significantly higher than the respective activities determined by the DPPH method (p < 0.001).

Figure 5 shows the antioxidant potential of Pinus mugo alcoholic extracts evaluated using the ABTS method. The higher activity was usually observed for extracts in diluted alcohols—it decreased with the prolongation of the extraction time. Moreover, the antioxidant activity of extracts in undiluted alcohols decrease with the extension of extraction time. The highest activity of 56.45 ± 2.37 TEAC was found for extracts in 40% methanol obtained after 15 min of extraction and for 40% ethanol after the same extraction time (53.60 ± 0.34 TEAC). The lower activity was found for extracts in undiluted alcohols; the lowest one was observed for extracts in undiluted n-propanol (2.76 ± 1.58 TEAC) and in undiluted isopropanol (7.42 ± 1.07 TEAC), both after 60 min of extraction. Similarly to Pinus strobus, the activity of the antioxidant potential evaluated with ABTS method was statistically significantly higher than the respective values obtained with the DPPH method (p < 0.001). Additionally, a statistically significant correlation of r = 0.8092 was observed between these parameters (p < 0.001). Moreover, the activities of Pinus mugo alcoholic extracts evaluated with the ABTS method were statistically significantly higher than those of Pinus strobus (p < 0.001).

Figure 6 presents the antioxidant activity of Pinus nigra alcoholic extracts determined using the ABTS method. The higher activity was observed for extracts in diluted alcohols—they gradually increased with the extension of the extraction time. The highest activity of 12.83 ± 1.05 TEAC was found for extracts in 40% methanol obtained after 60 min of extraction and for 40% ethanol after the same extraction time (11.88 ± 0.72 TEAC). The lower activity was found for extracts in undiluted alcohols; no activity was observed for isopropanol extract after 60 min of extraction; and very low activity was observed in the same solvent after the other extraction time and in undiluted ethanol (1.30 ± 0.13 TEAC) after 15 min of extraction. Similarly to Pinus strobus, the antioxidant potential evaluated using the ABTS method was statistically significantly higher than the respective values obtained using the DPPH method (p < 0.001). Moreover, the activities of Pinus nigra alcoholic extracts evaluated with the ABTS method differ significantly from Pinus strobus and Pinus mugo (p < 0.001).

The polyphenol content in Pinus strobus extracts prepared in the studied short-chain alcohols is presented in Figure 7. In contrary to the antioxidant activity evaluated with both the applied methods, the extracts in undiluted alcohols, particularly in n-propanol and isopropanol, contain higher amounts of polyphenols. Moreover, the amount of this group of compounds increased in general with the extension of the extraction time. The highest activity was observed for extracts in undiluted n-propanol and undiluted isopropanol following 60 min of extraction, i.e., 15.37 ± 0.70 GAE (mg gallic acid/g raw material) and 10.96 ± 0.93 GAE. The lowest content of polyphenols was found in extracts in 40% methanol obtained after 30 min of extraction—1.03 ± 0.44 GAE.

Figure 8 presents the polyphenol content in Pinus mugo extracts in the studied short-chain alcohols. The higher content was observed for extracts in diluted alcohols—this parameter gradually decreased with the increase in alcohol concentration in solvents used for extract preparation. The highest polyphenol content, evaluated with the Folin–Ciocalteu method, was found for 40% ethanol and 40% methanol used as extractants after 30 min of extraction—15.76 ± 0.10 and 9.71 ± 1.59 GAE, respectively. The lowest content of polyphenols was found in extracts in undiluted isopropanol obtained after 15 min of extraction—1.47 ± 0.65 GAE and in undiluted ethanol after 60 min of extraction—1.88 ± 0.31 GAE. The polyphenol content in Pinus strobus and Pinus mugo extracts differs significantly (p < 0.001). A statistically significant (p < 0.001) linear relationship between the antioxidant activities of Pinus mugo extracts evaluated with both the DPPH and ABTS methods and the polyphenol content evaluated using the Folin–Ciocalteu (F-C) method were found; the correlation coefficient was r = 0.8202, r = 0.7148, and r = 0.6076 for ABTS vs. DPPH, F-C vs. DPPH, and F-C vs. ABTS, respectively.

Figure 9 presents the polyphenol content in Pinus nigra extracts in the studied alcohols. This parameter slightly increased with the prolongation of the extraction time. The highest polyphenol content, evaluated using the Folin–Ciocalteu method, was found for 40% ethanol and 40% methanol used as extractants after 60 and 30 min of extraction time, respectively—6.60 ± 0.65 and 5.26 ± 0.34 GAE, respectively. The lowest content of polyphenols was found in extracts in undiluted n-propanol obtained after 30 min of extraction—0.64 ± 0.07 GAE and in undiluted isopropanol after 15 min of extraction—0.76 ± 0.44 GAE. The polyphenol content in Pinus nigra and Pinus mugo extracts differs significantly (p < 0.001). A statistically significant (p < 0.001) linear relationship between the antioxidant activities of Pinus nigra extracts evaluated with both the DPPH and ABTS methods and the polyphenol content evaluated with the Folin–Ciocalteu (F-C) method were found; the correlation coefficient was r = 0.9148, r = 0.7215, and r = 0.6843 for ABTS vs. DPPH, F-C vs. DPPH, and F-C vs. ABTS, respectively

In the Supplementary Materials, the graphs presented the comparison of the antioxidant potential of all extracts evaluated with the DPPH (Figure S1) and the ABTS methods (Figure S2); in addition, the polyphenol content in all extracts (Figure S3) are included.

In

Table 2. Parameters of photoprotection evaluated in vitro for Pinus strobus alcoholic extracts.

Solvent	Time	SPF		UVA/UVB		Critical Wavelength
		Mean	SD	Mean	SD
40% methanol	15 min	2.408	0.153	0.380	0.010	295
70% methanol	15 min	3.006	0.029	0.409	0.004	295
99% methanol	15 min	1.273	0.111	0.441	0.008	295
40% ethanol	15 min	3.212	0.025	0.402	0.002	295
70% ethanol	15 min	1.813	0.020	0.422	0.001	295
96% ethanol	15 min	1.323	0.022	0.488	0.003	296
40% n-propanol	15 min	1.700	0.021	0.379	0.002	295
70% n-propanol	15 min	1.020	0.036	0.395	0.008	295
99% n-propanol	15 min	0.794	0.193	0.425	0.010	295
40% isopropanol	15 min	1.568	0.020	0.402	0.005	295
70% isopropanol	15 min	1.200	0.017	0.427	0.004	295
99% isopropanol	15 min	0.466	0.006	0.489	0.009	296
40% methanol	30 min	1.133	0.027	0.349	0.001	295
70% methanol	30 min	1.620	0.017	0.382	0.006	295
99% methanol	30 min	1.644	0.039	0.448	0.011	295
40% ethanol	30 min	3.098	0.071	0.409	0.040	295
70% ethanol	30 min	2.834	0.034	0.433	0.003	295
96% ethanol	30 min	0.849	0.024	0.480	0.008	296
40% n-propanol	30 min	2.522	0.118	0.414	0.009	295
70% n-propanol	30 min	1.492	0.040	0.416	0.008	295
99% n-propanol	30 min	0.624	0.009	0.445	0.006	295
40% isopropanol	30 min	1.915	0.051	0.403	0.003	295
70% isopropanol	30 min	1.452	0.030	0.457	0.009	296
99% isopropanol	30 min	0.609	0.064	0.461	0.027	296
40% methanol	60 min	2.138	0.049	0.382	0.002	295
70% methanol	60 min	2.646	0.159	0.414	0.006	295
99% methanol	60 min	3.299	0.027	0.485	0.003	295
40% ethanol	60 min	2.380	0.038	0.408	0.006	295
70% ethanol	60 min	1.851	0.040	0.432	0.004	295
96% ethanol	60 min	1.093	0.095	0.452	0.011	296
40% n-propanol	60 min	2.417	0.015	0.384	0.003	295
70% n-propanol	60 min	2.182	0.025	0.432	0.006	295
99% n-propanol	60 min	1.183	0.043	0.463	0.005	296
40% isopropanol	60 min	2.371	0.152	0.427	0.010	295
70% isopropanol	60 min	1.683	0.036	0.448	0.013	296
99% isopropanol	60 min	0.974	0.047	0.500	0.023	296

,

Table 3. Parameters of photoprotection evaluated in vitro for Pinus nigra alcoholic extracts.

Solvent	Time	SPF		UVA/UVB		Critical Wavelength
		Mean	SD	Mean	SD
40% methanol	15 min	1.084	0.490	0.314	0.011	294
70% methanol	15 min	1.386	0.015	0.403	0.003	294
99% methanol	15 min	0.854	0.031	0.572	0.019	296
40% ethanol	15 min	1.607	0.057	0.384	0.003	295
70% ethanol	15 min	0.987	0.121	0.464	0.026	295
96% ethanol	15 min	1.304	0.017	0.467	0.004	294
40% n-propanol	15 min	2.260	0.063	0.341	0.004	294
70% n-propanol	15 min	1.703	0.036	0.403	0.003	294
99% n-propanol	15 min	1.325	0.063	0.500	0.017	295
40% isopropanol	15 min	1.492	0.071	0.278	0.006	293
70% isopropanol	15 min	1.514	0.041	0.393	0.006	294
99% isopropanol	15 min	0.932	0.124	0.449	0.044	294
40% methanol	30 min	2.316	0.456	0.241	0.007	293
70% methanol	30 min	2.188	0.046	0.303	0.004	293
99% methanol	30 min	2.356	0.028	0.464	0.003	295
40% ethanol	30 min	2.539	0.035	0.312	0.003	294
70% ethanol	30 min	2.338	0.116	0.369	0.007	294
96% ethanol	30 min	1.207	0.021	0.459	0.012	294
40% n-propanol	30 min	2.630	0.194	0.375	0.016	294
70% n-propanol	30 min	2.417	0.284	0.426	0.032	294
99% n-propanol	30 min	1.171	0.073	0.568	0.039	295
40% isopropanol	30 min	2.857	0.332	0.340	0.021	294
70% isopropanol	30 min	2.672	0.052	0.435	0.007	295
99% isopropanol	30 min	1.257	0.062	0.484	0.013	294
40% methanol	60 min	3.195	0.030	0.281	0.005	293
70% methanol	60 min	2.386	0.070	0.309	0.009	294
99% methanol	60 min	2.560	0.163	0.472	0.012	295
40% ethanol	60 min	3.268	0.086	0.326	0.007	294
70% ethanol	60 min	2.459	0.090	0.372	0.009	294
96% ethanol	60 min	1.524	0.076	0.502	0.009	295
40% n-propanol	60 min	3.403	0.071	0.393	0.005	294
70% n-propanol	60 min	2.351	0.459	0.421	0.004	294
99% n-propanol	60 min	1.425	0.046	0.527	0.015	295
40% isopropanol	60 min	2.660	0.111	0.353	0.003	294
70% isopropanol	60 min	1.947	0.114	0.423	0.015	294
99% isopropanol	60 min	1.662	0.039	0.525	0.027	295

and

Table 4. Parameters of photoprotection evaluated in vitro for Pinus mugo alcoholic extracts.

Solvent	Time	SPF		UVA/UVB		Critical Wavelength
		Mean	SD	Mean	SD
40% methanol	15 min	6.718	0.020	0.438	0.047	295
70% methanol	15 min	3.662	0.074	0.407	0.006	295
99% methanol	15 min	2.664	0.090	0.479	0.003	295
40% ethanol	15 min	4.663	0.271	0.438	0.047	295
70% ethanol	15 min	2.250	0.330	0.450	0.045	295
96% ethanol	15 min	0.942	0.287	0.461	0.002	295
40% n-propanol	15 min	2.664	1.587	0.423	0.002	295
70% n-propanol	15 min	2.605	0.126	0.429	0.003	295
99% n-propanol	15 min	1.070	0.030	0.556	0.006	296
40% isopropanol	15 min	2.812	0.076	0.413	0.006	295
70% isopropanol	15 min	1.775	0.187	0.413	0.021	295
99% isopropanol	15 min	0.460	0.031	0.540	0.036	296
40% methanol	30 min	4.356	0.040	0.381	0.006	295
70% methanol	30 min	2.522	0.097	0.367	0.001	295
99% methanol	30 min	1.730	0.091	0.464	0.005	295
40% ethanol	30 min	2.605	0.075	0.385	0.005	295
70% ethanol	30 min	2.652	0.119	0.418	0.029	295
96% ethanol	30 min	0.966	0.041	0.480	0.008	296
40% n-propanol	30 min	5.206	0.094	0.454	0.006	295
70% n-propanol	30 min	2.408	0.038	0.445	0.003	295
99% n-propanol	30 min	0.730	0.098	0.506	0.024	296
40% isopropanol	30 min	1.215	0.772	0.415	0.001	295
70% isopropanol	30 min	1.039	0.084	0.404	0.011	295
99% isopropanol	30 min	0.462	0.040	0.477	0.025	296
40% methanol	60 min	3.743	0.211	0.407	0.009	295
70% methanol	60 min	4.214	0.229	0.465	0.032	295
99% methanol	60 min	2.710	0.097	0.517	0.006	296
40% ethanol	60 min	4.431	0.035	0.423	0.001	295
70% ethanol	60 min	2.996	0.104	0.451	0.004	295
96% ethanol	60 min	1.545	0.040	0.455	0.004	295
40% n-propanol	60 min	4.098	1.507	0.447	0.005	295
70% n-propanol	60 min	2.895	0.059	0.449	0.002	295
99% n-propanol	60 min	0.767	0.210	0.532	0.016	296
40% isopropanol	60 min	3.930	0.660	0.428	0.016	295
70% isopropanol	60 min	3.096	0.079	0.446	0.003	295
99% isopropanol	60 min	1.244	0.020	0.485	0.007	296

, the parameters of photoprotection applied for the preliminary in vitro evaluation of the studied extracts of Pinus strobus, Pinus mugo, and Pinus nigra are summarized, depending on the applied solvent for the ultrasound-assisted extraction and time of extraction. As the absorbance of the extracts was high, to evaluate these parameters, samples were diluted more than 25 times as described in Material and Methods.

The sun protection factor (SPF) of diluted alcoholic extracts of Pinus strobus varied from 0.466 ± 0.006 in 99% isopropanol after 15 min of extraction to 3.299 ± 0.027 in undiluted methanol after one hour of extraction. It can be seen that, in general, higher values of SPF were found for Pinus strobus extracts in more diluted alcohols, except for those in methanol, for which an inverse relationship was observed. Regarding other parameters, the UVA/UVB ratio indicating protection against UVA as compared to UVB varied from 0.349 ± 0.01 for extracts in 40% methanol after 30 min of extraction to 0.500 ± 0.023 in undiluted isopropanol after 60 min of extraction. The values below 0.4 described the minimal protection category, whereas, from 0.4 to 0.6, they are considered as among the moderate UVA protection category. Unfortunately, in the case of the studied raw materials, the SPF values of the extracts in undiluted alcohols were the lowest—for undiluted isopropanol used as a solvent after one hour of extraction, it was 0.794 ± 0.048. Another evaluated parameter was the critical wavelength—for all the extracts, it varied between 295 and 296 nm. This value confirmed that the protection against UVA was low (Table 2). Moreover, a statistically significant linear relationship of SPF values determined for all the studied extracts of Pinus strobus versus antioxidant activity determined either by the DPPH or ABTS methods (correlation coefficients were 0.7903 and 0.9356, respectively) was found (Figure 10). No statistically significant correlation between the polyphenol content vs. SPF and between the polyphenol content vs. antioxidant activity determined with either the DPPH or ABTS method was observed for Pinus strobus extracts.

The sun protection factor (SPF) for diluted alcoholic extracts of Pinus nigra varied from 0.854 ± 0.031 in 99% methanol after 15 min of extraction to 3.403 ± 0.071 in 40% n-propanol after one hour of extraction. Similarly to Pinus strobus, it can be seen that, in general, higher values of SPF was found for extracts in more diluted alcohols used as extractants. Regarding other parameters, the UVA/UVB ratio, indicating protection against UVA as compared to UVB, varied from 0.241 ± 0.007 for extracts in 40% methanol after 30 min of extraction, to 0.568 ± 0.039 in undiluted isopropanol after 30 min of extraction. The protection against UVA of this last extract can be considered as moderate. Unfortunately, in the case of the studied raw materials, the SPF values of the extracts in undiluted alcohols were the lowest, for instance, 1.171 ± 0.073 for the above-mentioned extract in undiluted isopropanol. Another evaluated parameter was the critical wavelength—for all the extracts, it varied between 293 and 295 nm. This value is similar to the respective values of other extracts prepared from all the studied raw material. Moreover, this observation confirmed that the protection against UVA was low (Table 3). Additionally, a statistically significant linear relationship of SPF values determined for all the studied extracts of Pinus nigra vs. antioxidant activity determined either by the DPPH or ABTS methods (correlation coefficients were 0.7903 and 0.9356, respectively) was found (Figure 11). A significant (p < 0.001) linear relationship between the polyphenol content (F-C method) and SPF (r = 0.7658) was also observed (Figure 11).

The sun protection factor (SPF) for diluted alcoholic extracts of Pinus mugo varied from 0.460 ± 0.031 in 99% isopropanol to 6.718 ± 0.020 in 40% methanol, both after 15 min of extraction. In general, higher SPF values was found for extracts in more diluted alcohols used as extractants. Regarding other parameters, the UVA/UVB ratio, indicating protection against UVA as compared to UVB, varied from 0.367 ± 0.001 for extracts in 70% methanol after 30 min of extraction, to 0.556 ± 0.006 in undiluted isopropanol after 15 of min extraction. The protection against UVA of this last extract can be considered as moderate. Unfortunately, in the case of the studied raw materials, the SPF values of the extracts in undiluted alcohols were the lowest, for instance, 1.070 ± 0.030 for the above-mentioned extract in undiluted isopropanol. Another evaluated parameter was the critical wavelength—for all the extracts, it varied between 295 and 296 nm. This value is similar to the respective values of other extracts prepared from all the studied raw material. This observation confirmed that the protection against UVA was low (Table 4). Additionally, a statistically significant linear relationship of SPF values determined for all the studied extracts of Pinus mugo vs. antioxidant activity determined either by the DPPH or ABTS methods was seen (correlation coefficients were 0.7948 and 0.8437, respectively) (Figure 12). A significant (p < 0.001) linear relationship between the polyphenol content (F-C method) and SPF (r = 0.6672) was also found (Figure 12). Figure S4 in the Supplementary Materials presents the comparison of the SPF parameters determined in vitro of all studied extracts of Pinus strobus, Pinus nigra, and Pinis mugo.

4. Discussion

People pay attention to their appearance, to ensure better well-being and better acceptance by others. They are looking for products containing antioxidants, i.e., ingredients that have beneficial effects on appearance.

In order to meet consumer expectations, raw materials, especially those of natural origin, are sought. Such products should demonstrate antioxidant activity, and delay the aging process of the body. Due to the constant exposure of our body to UV radiation, it would be important to find a raw material that will exhibit both photoprotective and antioxidant activity [49,50]. The combination of sunscreens with antioxidant agents may also be helpful for decreasing the risk of skin cancer or other skin damage caused by UV radiation [51,52].

This type of study seems to be helpful in developing new anti-aging cosmetics. Such studies are scarce; however, recently, some papers on an in vitro comparison of the antioxidant potential and sun protection factor of different plants have been published [52,53,54,55,56]. Therefore, we decided to compare the antioxidant activity and photoprotective parameters in vitro of extracts from three selected pine species, i.e., Pinus strobus, Pinus nigra, and Pinus mugo. To prepare the extracts, four short-chain aliphatic alcohols, namely, methanol, ethanol, n-propanol, and isopropanol, were applied, each at three concentration: 40% (v/v), 70% (v/v), and concentrated. Ultrasound-assisted extraction for 15, 30, or 60 min was used. This method belongs to the green extraction techniques due to the low energy consumption and high efficiency [57,58,59].

In the present study, the higher antioxidant activity determined by the DPPH method was found for extracts prepared in diluted alcohols. This was observed for all pines evaluated in the study. The highest activity among the Pinus strobus extracts was found for extracts in 40% methanol after one hour of extraction. The highest antioxidant potential evaluated by the ABTS method was also found for extracts in the same extractant, but after 15 min of extraction—these values were significantly higher than those determined by the DPPH method. Moreover, a significant correlation between the results obtained by these two methods was found (r = 0.81). With regard to the SPF, this parameter was higher for extracts in diluted alcohols except methanol—for this alcohol used as extractant, the highest value was found for the extract in undiluted methanol after 60 min of extraction. It should be added that a statistically significant correlation was found between the SPF and activity determined with either the DPPH (r = 0.94) or ABTS method (r = 0.79).

Liu at al. [60] evaluated the antioxidant potential of essential oil isolated from the needles of ten Pinus taxa by steam distillation. They also found higher activity obtained by the ABTS method as compared with the DPPH one. A statistically significant correlation between these results was also observed (r = 0.66) [60]. Kang and Howard evaluated the total phenolic content in the extract of, among others, Pinus strobus needles in 80% acetone [61]. They found that P. strobus extract contained more total phenols of 157 ± 2 mg gallic acid/ g powder (dry extract), and this value was higher compared with P. pinea, P. koraiensis, P. silvestris, and P. densiflora [61].

In our study on Pinus nigra extracts in short-chain alcohols, the higher antioxidant activity was found for extracts in diluted alcohols; however, this parameter, evaluated either with the DPPH or ABTS methods, slightly increased with the extension of the extraction time. This activity evaluated either with the DPPH or ABTS method was the highest for extract in 40% methanol after the one-hour ultrasound-assisted extraction. The results obtained with the ABTS method were statistically significantly higher than those determined with the DPPH technique (p < 0.001). A significant correlation between the activities evaluated with both methods (r = 0.91) was found. Moreover, contrary to P. strobus, statistically significant correlations between the polyphenol content and antioxidant potential of the extracts evaluated with the DPPH and ABTS methods were found (r = 0.72 and 0.68, respectively; p < 0.001). With regard to the SPF, these values were higher for the extracts of P. nigra in diluted alcohols, and, in general, the highest SPF was found for extracts in 40% alcohols. A statistically significant correlation was observed between the SPF and antioxidant activity evaluated with both the DPPH and ABTS method, as well as with the polyphenol content (r = 0.83, 0.85, and 0.76, respectively).

Fkiri et al. evaluated ethanol extracts obtained from 19 samples of Pinus nigra [21]. The ratio of raw material to solvent was the same as in our study, but the needles were extracted by continuous shaking for 24 h. The mean phenol content varied between 15.7 and 47.5 mg GAE/g and was higher than in our study. The antioxidant activity evaluated by the DPPH method varied from 36 to 93%. These values were higher than observed in our study, although the lowest activity was comparable [21].

Koutsaviti et al. evaluated the antioxidant potential of hydroxyethanolic extracts of Pinus nigra, Pinus strobus, and Pinus mugo needles using peroxy-oxalate and luminol chemiluminescence methods [14]. They found a similar mean antioxidant potential of the examined extracts of the studied needles [14].

Nisca et al. evaluated Pinus nigra bark extracts in 70% ethanol obtained with ultrasound-assisted extraction for 30 min using the same ratio of raw material to the solvent to prepare the extracts [23]. The antioxidant activity evaluated by the DPPH and ABTS methods was presented as per cent of inhibition. The antioxidant potential determined by the DPPH method was lower compared with our results (expressed as % of inhibition), whereas the opposite results were obtained with the ABTS method. With regard to the polyphenol content, this parameter was markedly higher in bark extracts obtained by Nisca et al. compared to the needle extract in the same solvent [23].

The antioxidant activity of the Pinus mugo needle extracts evaluated in the present study was significantly higher than those of P. strobus and P. nigra (p < 0.001). Similarly to the Pinus strobus extracts, the higher activity evaluated with the DPPH method was observed for extracts in diluted alcohols—they were comparable in the same solvents regardless of the extraction time. The highest activity was found for 40% ethanol used as a solvent after 15 min of extraction and for 40% methanol after 30 min of extraction. If the ABTS method was applied, the antioxidant activity of extracts in undiluted alcohols decreased with the extension of extraction time. In addition, the antioxidant potential evaluated with the ABTS method was statistically significantly higher than the activities obtained with the DPPH method (p < 0.001). A statistically significant correlation of r = 0.82 was observed between these parameters (p < 0.001), as well as between the SPF values vs. antioxidant activity determined either by the DPPH or ABTS methods (correlation coefficients were 0.79 and 0.84, respectively) (Figure 12). A significant (p < 0.001) linear relationship between the polyphenol content (F-C method) and SPF (r = 0.6672) was also found. The studied extracts were effective in photoprotection against UVB, while, in most cases, their effectiveness in the UVA range based on the UVA/UVB ratio was low.

Karapandzova et al. evaluated the antioxidant activity and total polyphenol content in Pinus mugo needle extracts prepared in 70% methanol using ultrasound-assisted extraction for 30 min [62]. They studied samples collected in two consecutive years from plants growing at two altitudes (2039 and 1640 m) in Macedonia. The total polyphenol content was between 11.4 and 12.2 mg gallic acid/g. This value was comparable with our results—5.67 mg gallic acid/g raw material, but our extracts were twofold-diluted compared with those of Karapandzova et al. They also determined the antioxidant potential of their extracts using the DPPH method and their IC₅₀ varied from 11.6 to 16.0 mg/mL; the higher values was found for plants growing at lower altitudes [62].

Popescu et al. determined the antioxidant activity of Pinus mugo needle extracts in 80% methanol [29]. Needles were collected from three mountain areas in Romania. After the samples were refluxed three times, the dry extract was obtained. For further analysis, dry extracts at a concentration of 5 mg/mL were applied. The antioxidant activity evaluated by the DPPH method varied from 124.3 to 165.0 mM trolox/g dry extract, whereas the total polyphenol content varied from 55.5 to 68.2 mg gallic acid/g extract [29].

5. Conclusions

Based on the obtained results, it can be concluded that the studied extracts of Pinus strobus, P. nigra and P. mugo have antioxidant potential. The extracts obtained using diluted alcohols for ultrasound-assisted extraction were the most favorable. According to the correlation analysis, there is a significant positive correlation between antioxidant activity and the evaluated in vitro sun protection factor of the tested extracts. However, the studied extracts were effective against UVB, while, in most cases, their effectiveness in the UVA range was low. Comparing the tested pines, the best results were obtained for Pinus mugo. The use of such raw materials due to their antioxidant and photoprotective properties could be taken into account in the development of new cosmetic preparations. The current research is preliminary. In the next stages, we intend to expand their scope by using either other solvent concentrations or other solvents, as well as other extraction techniques to increase the activity of the obtained extracts, which could be useful for making new antiaging cosmetics.

Further studies should be carried out to identify chemical compounds in individual extracts, which will be helpful for improving the antioxidative and photoprotective potential of the studied extracts. Moreover, some other experiments, including, for instance, toxicological tests, should be performed to evaluate the safety of such preparations.