3.1. Characterization of Sweet Cherry Stem Extracts by HPLC-ESI-QTOF-MS
The scPLE, scSFE, and scSWE extracts were fully characterized by HPLC-ESI-QTOF-MS. For this analysis, the dried extracts were reconstituted in EtOH, EtOH-H2
O (50:50), and H2
O, respectively, up to a concentration of 1000 mg/L. Their chromatographs are shown in Figure 1
The compounds were tentatively identified using the information provided by the software (accurate masses, isotopic distributions, MS spectra, and molecular formula), together with the fragmentation patterns obtained from tandem mass spectrometry (MS/MS) experiments in comparison with standards when available or data previously reported in the literature. A total of 57 compounds were identified from 4 different families: (1) organic acids, phenolic acids, and derivatives (8 compounds); (2) flavonoids and derivatives (36 compounds); (3) fatty acid derivatives (9 compounds); and (4) terpenes (4 compounds). Thus, 18 of these compounds are herein identified for the first time in this matrix. The identities of the obtained compounds are summarized in Table 1
, and these results significantly advance and complete the previous data available on these extracts obtained using both gas chromatography coupled to mass spectrometry (GC-MS) [21
] and HPLC-ESI-QTOF-MS [19
Semiquantitative comparisons among the different extraction techniques regarding the presence of individual compounds in those extracts can be observed in Table 2
. This information is useful for determining which technique is better for extracting particular types of compounds. In general, as depicted in Figure 2
, organic and phenolic acids and derivatives are present at higher concentrations in the extract obtained by SWE (scSWE). In addition, flavonoids were more abundant in the PLE extract (scPLE); as expected, fatty acid derivatives and terpenes, which are nonpolar in nature, were better extracted by SFE (scSFE). Similar results have been reported for other natural compounds, such as marine compounds [31
] and polyphenols [32
3.2. Total Phenolic Contents and Antioxidant Capacities of the SC Stem Extracts
After the analytical processing of the extracts, a multistep screening of the three extracts was conducted in three stages to select the best extract with the greatest potential as a novel cosmetic ingredient (see graphical abstract). This screening was designed to evaluate the most relevant biological activities for the cosmetic industry using a collection of in vitro and cellular assays, all of which are described in the Materials and Methods section.
The first step evaluated the total phenolic content (TPC). This assay was the first step because polyphenolic compounds are one of the main groups of compounds known to show anti-aging effects and present other biological activities [33
]. Furthermore, higher polyphenolic contents typically result in more intense biological activities [35
]. The TPC was evaluated using the Folin-Ciocalteu assay, as described in the Materials and Methods Section. In addition to TPC measurement, this first stage included the TEAC assay, which is accepted as a general method for measuring antioxidant activity. The relationship between the total polyphenolic content and the antioxidant activity has been demonstrated previously by numerous studies published by our group [25
] and others [35
The results for this first stage are presented in Table 3
. Among the SC stem extracts, scPLE and scSFE had the highest total polyphenolic contents, and their contents were not significantly different (p
> 0.05). scSFE showed the highest antioxidant effect in the TEAC assay, followed by scPLE. However, scSWE showed a remarkably lower TPC and antioxidant capacity (at least p
< 0.001 and p
< 0.0001, respectively). Thus, both assays showed similar trends for the three extracts, with the lowest antioxidant activity for that extract with the lowest polyphenolic content, as expected. On the basis of these results, scSWE was not included in for further screening steps due to its poorer results.
In a second stage, additional antioxidant assays, FRAP and ORAC, were carried out to clarify the antioxidant activities of the selected extracts (scSFE and scPLE). FRAP estimates the Fe(III) reducing activity, whereas the ORAC assay determines the activity related to chain-breaking antioxidants, which is directly related to peroxyl radicals. These analyses are more closely related to the biological function of antioxidants [25
]. The results are shown in Table 4
. Both extracts presented significant antioxidant activities with higher values for scSFE in the ORAC assay and for scPLE in the FRAP assay. These values are higher than those of other extracts from Cistus
sp. plants obtained by aqueous and hydroalcoholic conventional extraction methods previously characterized by our group [25
Although scSFE and scPLE presented some differences, probably due to differences in their composition, as shown in Section 3.1
, both exhibited high antioxidant activities that deserve further investigation. The higher potency of scPLE compared with scSFE in the FRAP assay was probably due to scPLE’s higher content of flavonoids bearing a catechol group in their B ring, such as catechins and quercetin derivatives, which can complex metal ions. However, the results for scSFE were more interesting from a cosmetic point of view, as ORAC indicates the capacity to scavenge peroxyl free radicals and other radicals derived from lipid peroxidation, and these radicals are frequent in cosmetic products due to the inclusion of oily ingredients in their formulation. Furthermore, the SFE extraction technique is more suitable for scale-up for use at large industrial facilities than is PLE, which is a less developed extraction technique at the industrial level. For these reasons, scSFE was selected for full characterization in the further assays included in the third stage of the screening.
The third stage further elucidated the antioxidant capacity of scSFE through additional antioxidant assays. In this sense, TBARS for the specific study of lipid peroxidation, a modified ORAC method based on OH·
radicals, and a Griess nitrite-based assay for nitric oxide radicals were performed. The results for all these assays are shown in Table 5
scSFE showed a significant antioxidant capacity in all the assays, suggesting that this extract is a good candidate for use as a bioactive ingredient against oxidative stress, as the extract has shown antioxidant capacity through different methods and against different targets, such as lipid peroxidation and different kinds of free radicals.
Antioxidant activities are highly desirable for cosmetic ingredients for several reasons. On the one hand, this activity protects the final formula itself from oxidation, especially from oxidation related to its oily ingredients. In addition to this advantage, which is mainly related to the final product formulation, the antioxidant activity of the ingredients is probably one of the most commonly used claims in cosmetic products. In this sense, natural extracts have been shown to reduce oxidative stress, mainly due to their polyphenols [40
]. scSFE contains different families of compounds, as shown in Figure 2
, and polyphenols (including flavonoids, organic acids, phenolic acids, and their derivatives) were the most abundant. Thus, the antioxidant effects of scSFE could be mainly due to the polyphenolic compounds present in this extract, but contributions from other compounds, especially terpenes, cannot be discarded. Catechins, naringenin, and chrysin are the main polyphenols in this extract, and thus the reduction in lipid peroxidation and the depletion of hydroxyl radicals and nitric oxide could have been due to these compounds, which have been demonstrated to have antioxidant capacities. Naringenin is a flavanone, catechin is a flavanol, and chrisin is a flavone. Compounds of these types have been shown to have antioxidant activities. Naringenin, which is found in citrus fruits, grapes, and other fruits, has shown antioxidant effects through lipid peroxidation reduction; increases in antioxidant defense; and scavenging free radicals, such as hydroxyl, superoxide, hydrogen peroxide, and nitric oxide radicals [42
]. Moreover, catechins have been shown to have antioxidant activity through many assays, such as the ABTS and FRAP assays. They protect against AAPH-induced peroxide radicals and lipid peroxidation and can scavenge free radicals [44
]. In addition to naringenin and catechins, chrysin and its derivatives reduce lipid peroxidation, regulate redox homeostasis, and increase antioxidant enzymes [46
]. The antioxidant effects of these compounds are related to the carbonyl group at C-4 and the double bond between C2 and C3 [48
3.3. Skin Aging-Related Enzymatic Assays
In this third stage, the putative modulative activities of scSFE on some of the most relevant enzymes related to skin health and appearance were also tested. In this sense, the activities of collagenase, elastase, hyaluronidase, and tyrosinase were challenged with scSFE, as detailed in the Materials and Methods section. These experiments were concluded with a study of the inhibition of advanced glycosylation end product (AGE) formation, conducted as described in the Materials and Methods section. The inhibition of collagenase, tyrosinase, elastase, hyaluronidase, and AGE formation are related to the prevention of the degradation of extracellular matrix (ECM), skin preservation, and antiaging. In fact, plant extracts have been shown to inhibit tyrosinase, collagenase, elastase, and hyaluronidase activity [49
]. The results, shown in Table 6
, are expressed as the percentage of inhibition for each assay.
Collagen plays a critical role in the appearance and function of the skin; it confers tensile strength and resiliency to the skin and is the main protein in the ECM of the dermis. Its degradation is related to skin wrinkling and aging. Collagenase inhibition is related to the maintenance of skin tensile strength and elasticity, even more so in collagenase induction by ROS or irradiation, which are important factors in aging. In this case, scSFE did not present any collagenase inhibition activity, presenting a negative value, which means that its effect was weaker than that of the negative control but was not statistically significant.
Tyrosinase is an enzyme involved in melanin production, the main defense of organisms against UV irradiation. Melanin absorbs UV radiation and reduces the formation of photoproducts that could be harmful to the skin [52
]. Tyrosinase induction is related to skin protection through an increase in melanin production, and tyrosinase inhibition could be useful in diseases such as vitiligo. As shown in Table 6
, scSFE showed a moderate but not statistically significant effect compared with the untreated negative control.
Elastin is an extracellular matrix protein responsible for elasticity in the dermis and other connective tissues by forming elastin fibers. Elastase is an enzyme able to degrade elastin, leading to skin aging and wrinkles. Therefore, the inhibition of elastase is related to skin aging and wrinkle protection. The results in Table 6
indicate that scSFE showed potent elastase inhibition activity, even above the PMSF positive control, and that the effect was significant (** p
Hyaluronic acid is found in connective tissue and is part of the ECM. Hyaluronic acid presents water holding properties and maintains the viscosity and the correct permeability of connective tissues and maintains skin hydration. Hyaluronidase degrades hyaluronic acid, and its inhibition is related to the maintenance of high levels of hyaluronic acid, improving the general aspect of skin and specifically skin hydration. scSFE presented potent hyaluronidase inhibition activity, reaching almost 100% of the level obtained for p-dimethylaminobenzaldehyde (positive control), and the effect was highly significant (**** p < 0.0001).
Finally, oxidative stress increases protein glycation, which is responsible for advanced glycosylation end products (AGEs) in skin. AGEs are one of the causes of collagen degradation, leading to skin aging. The inhibition of protein glycation is related to the prevention of aging and wrinkling. As expected by its antioxidant capacity shown in the previous section, scSFE was able to reduce AGE formation by 50% with high statistical significance (**** p < 0.0001).
A wide variety of phytomolecules belonging to different classes of polyphenols, terpenoids, or steroids (e.g., catechins, carnosic acid, ellagic acid, curcumin, and hydroxycinnamic acids) are inhibitors of collagenase, elastase, and hyaluronidase [53
]. Some plant extracts containing these compounds scavenge free radicals, mainly due to polyphenols, protecting the skin matrix through the inhibition of enzymatic degradation and/or promoting the synthesis of its components, improving skin elasticity and tightness [57
]. The polyphenols present in scSFE could be responsible for its ability to inhibit cosmetic enzymes. As shown in [59
], catechin and epigallocatechin gallate inhibit collagenase and elastase, and naringenin inhibits hyaluronidase. This activity is related to the number of hydroxyl groups, as more available hydroxyl groups result in higher activity, and the inhibition of these enzymes decreases with substitution of hydroxyl groups or glycosylation [60
]. Furthermore, an extract of Libidibia ferrea
, whose main constituents are ellagic acid, catechin, and epicatechin, inhibited elastase, hyaluronidase, and tyrosinase, but presented a weak inhibition of collagenase, similar to what is seen with scSFE [61
]. These results may suggest that catechins, which are the main components of scSFE, could be responsible for the activity observed in these cosmetic assays. Further studies must be conducted to identify the molecules related to each inhibition activity, as well as their inhibition mechanisms, as it is documented that natural compounds can interact with these enzymes through different methods, such as competitive and/or noncompetitive inhibition [59
The overproduction or accumulation of melanin could lead to pigmentary disorders such as vitiligo, and it is related to skin aging and photoprotection. Tyrosinase is the enzyme that regulates the hydroxylation of L-tyrosine to form 3,4-Dihydroxy-L-phenylalanine (L-DOPA), a precursor of melanin. Inhibiting tyrosinase is a method for avoiding disorders related to skin hyperpigmentation, and in vitro enzymatic assays, as employed in the present study, are significantly related to melanin synthesis in melanocytes [63
]. Some polyphenols obtained from plants can inhibit tyrosinase and melanogenesis. In fact, catechin and its derivates, such as those present in scSFE, potently inhibit tyrosinase, and thus these flavanols could be the main flavanols responsible for the tyrosinase inhibition shown by scSFE. In addition, some polyphenol mixtures, such as mixtures of glabridin and resveratrol, show a synergistic tyrosinase inhibition [64
]. A synergistic effect could increase the value of plant extracts such as scSFE, which are characterized by the presence of many different compounds. However, a synergistic approach similar to that described in [65
] must be developed after the identification of the products responsible for the tyrosinase inhibition and other biological activities.
3.4. scSFE Showed Photoprotection Activity against UVB Irradiation
As the last set in the third stage of the screening, the photoprotective effect of scSFE was evaluated in HaCaT cells. Viability after UVB irradiation (800 or 1200 J/m2
) was first determined through MTT assay in the presence of different concentrations of scSFE (Figure 3
scSFE extract increased the viability of cells 24 h after UVB irradiation compared to the irradiated control. At 800 J/m2
, 100 µg/mL scSFE extract (0.01% w/v
) increased cell viability compared to untreated irradiated cells, whereas 100 µg/mL extract did not protect against the 1200 J/m2
dose. This protective effect was greater in the highest treatment concentration (200 µg/mL, 0.02% w/v
) after 800 and 1200 J/m2
irradiation, with a statistically significant protective effect compared to the untreated control. The highest photoprotection activities were observed for 800 J/m2
, 14.61% with 100 µg/mL extract, and 36.53% photoprotection with 200 µg/mL extract. However, weaker effects were obtained after 1200 J/m2
of UVB irradiation, with 3.51% and 13.99% photoprotection, respectively, compared to control as shown in Table S1
. Similar results with other natural extracts, such as citrus, rosemary, and lemon balm extracts, have been obtained using the same technique, with protection levels ranging from 10% to 80% [22
This photoprotective activity of scSFE could be due to various factors. On the one hand, scSFE showed a significant absorption in the UV range in a dose-dependent manner, and thus a substantial portion of the observed keratinocyte photoprotection could be due to the ability of the compounds present in this extract to absorb and scavenge UVB radiation, as many plant extracts have been shown to do [23
]. On the other hand, intracellular mechanisms may be involved in scavenging UVB-induced free radicals, attenuating death mechanisms and/or DNA damage, as other plant extracts have been shown to do [23
]. In fact, some of the main compounds present in scSFE have photoprotective activities through different mechanisms. Naringenin has been shown to increase keratinocyte survival and inhibit apoptosis and pyrimidine dimers after UVB radiation [73
]. Epigallocatechin gallate reduces UVB-induced damage in keratinocytes [74
], and catechin may protect skin cells against UVB-induced damage through its antioxidant activity [76
]. In addition, chrysin has shown UVB protection activity by attenuating UVB-induced apoptosis, ROS generation, and cyclooxygenase 2 expression [77
]. All these data suggest that the photoprotective effects of scSFE could be due to the different activities of the polyphenols naringenin, catechin, chrysin, and their derivates, which are the main polyphenolic compounds present in scSFE.
3.5. scSFE Inhibited Intracellular ROS Generation Induced by UVA and UVB Light in HaCaT Cells
As mentioned in the previous section, one of the putative mechanisms involved in scSFE photoprotection may be its antioxidant properties. To check if the antioxidant properties shown by scSFE in the previous sections were also present at the cellular level, the antioxidant capacity of the extract was evaluated in vitro through the determination of intracellular ROS in the HaCaT cell line. Ultraviolet (UV) A and B were used to induce oxidative stress, as determined by measuring dichlorofluorescein-diacetate H2
DCF-DA fluorescence, as described in the methods section. Figure 4
shows ROS generation after UVA (3 or 6 J/cm2
) or UVB (800 or 1200 J/m2
) irradiation in the absence or presence of the extract compared to the control (without irradiation).
Oxidative stress was inhibited by all the concentrations at all the UV doses (both UVA and UVB) tested in this assay. The reduction of fluorescence observed in all conditions is displayed in Table S2
DCF-DA is a fluorescent probe that is particularly sensitive to H2
OH, and peroxynitrite radicals at the intracellular level [79
]. The ORACOH
assay showed that scSFE has a significant capacity to scavenge •
OH, a harmful radical that can be derived from the Fenton reaction of H2
or from lipid peroxides, and a significant capacity to eliminate NO•
radicals, which may form peroxynitrite upon reaction with O2•−
. Therefore, the photoprotective properties of scSFE shown in this study may be related to its capacity to decrease the generation of intracellular radical species such as H2
OH, or peroxynitrite, which can damage a wide range of molecules in cells, including proteins and DNA [23
]. The major polyphenolic compounds in scSFE, catechin, chrysin, and naringenin have previously shown antioxidant activity against these free radicals [42
], with concomitant antioxidant activity against UV-induced oxidative stress [44
], confirming these statements. Similar antioxidant and photoprotective effects from UVA and UVB radiations have been also documented for a well-known cosmetic ingredient, ascorbic acid [81
], reinforcing the putative potential of scSFE extract as a new cosmetic ingredient.