Several biological activities have been reported for leaf extracts of Cecropia pachystachya species, including antioxidant and wound healing activities. This study aims to report, for the first time, the antiaging potential of the hydroethanolic HE and the ethanolic EE extracts obtained from the leaves of C. Their ability to prevent the production of advanced glycation end products AGEs was also evaluated, and both extracts showed important activity, especially HE. The extracts also stimulated the fibroblasts proliferation in vitro , specialized cells that produce several mediators which maintain the skin integrity and youthfulness. Cytotoxicity of the extracts was not observed for this lineage or HEK, human embryonic kidney cells widely used to evaluate cytotoxicity of chemical compounds. HE also exhibited the ability to inhibit the collagenase metalloproteinase MMP-2 and elastase activities.
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Several biological activities have been reported for leaf extracts of Cecropia pachystachya species, including antioxidant and wound healing activities.
This study aims to report, for the first time, the antiaging potential of the hydroethanolic HE and the ethanolic EE extracts obtained from the leaves of C. Their ability to prevent the production of advanced glycation end products AGEs was also evaluated, and both extracts showed important activity, especially HE.
The extracts also stimulated the fibroblasts proliferation in vitro , specialized cells that produce several mediators which maintain the skin integrity and youthfulness. Cytotoxicity of the extracts was not observed for this lineage or HEK, human embryonic kidney cells widely used to evaluate cytotoxicity of chemical compounds. HE also exhibited the ability to inhibit the collagenase metalloproteinase MMP-2 and elastase activities.
The total phenolic and flavonoids contents were also determined. HPLC analysis revealed the presence of the flavonoids orientin and iso-orientin, which were quantified to be used as chemical markers.
The results suggested that the extracts of C. The normal functions and the natural appearance of the skin are severely affected by age and by many factors from the external environment which accelerate the skin aging, including ultraviolet radiation and pollution [ 1 ]. Nowadays, the search for new dermocosmetics capable of preventing and treating the skin aging process is gaining attention due to the increase in life expectancy and to the people concern in order to maintain a young appearance [ 2 ].
The harmful central mechanism that promotes skin aging is associated with the continuous solar ultraviolet radiation exposure which induces a complex and specific sequence of cellular responses, especially related to the synthesis of reactive oxygen species ROS , which lead to noxious stimulus to the connective skin tissue [ 3 ].
Oxidative stress is also capable of altering the regulation of cellular mediators associated with the increase of metalloproteases expression, which are enzymes responsible for the degradation of extracellular matrix constituents, including collagen and elastin [ 4 ]. Also, ROS may promote direct oxidative damage in several cellular components, including organelle, nucleic acids, and plasmatic and mitochondrial membranes [ 5 ].
It is noteworthy mentioning that some histological changes are consequences of protein modifications secondary to oxidative reactions, including glycation reaction. For instance, dicarbonyl compounds produced by oxidative stress can bind to dermal matrix proteins, as collagen and elastin, which leads to the production of advanced glycation end products AGEs. These chemical complexes may accumulate and accelerate the process of fibroblast apoptosis, aggravating the aging phenomenon [ 6 , 7 ].
There is considerable interest in searching for new cosmetic ingredients that can be used as antiaging agents, notably those derived from natural sources [ 8 ].
Several pharmacological studies have reported the biological activities for different C. For instance, the methanolic extract showed significant hypotensive and anti-inflammatory activities [ 11 , 12 ]. Besides, the ethyl acetate extract demonstrated strong wound healing effects [ 13 ].
It is worthy pointing out that both extracts showed significant antioxidant capacity [ 12 , 13 ]. As the antiaging potential of natural substances is at least in part related to their antioxidant activity, those results are particularly impressive, as they encourage novel studies aimed to identify new extracts capable of reducing the aging process. Also, Duque et al. Thus, the primary objective of this study was to assess the antiaging potential of C. Ethanol and a mixture of ethanol-water were used as solvents as they are more feasible for dermal application in human skin, due to the lack of toxicity.
All other reagents were of the highest quality available. The plant was identified by a botanical specialist. Both extracts were kept in tightly stoppered bottles under refrigeration until the experimental procedures.
The total phenolic content was determined by the Folin—Ciocalteu method with some modifications [ 15 ]. The phenolic content was calculated using a calibration curve of tannic acid standard solutions 0.
All measurements were performed in triplicate. The amount of flavonoids was determined by aluminum chloride reagent assay, as described by Dowd [ 17 ], with slight modifications. Briefly, aliquots of 0. Water was used as blank control.
An automatic injector and a Zorbax SB-C18 column 50 mm x 4. The mobile phase was consisted of a gradient elution of methanol: H 2 O, as described in Table 1. The flow rate used was 0. The total antioxidant activity was evaluated by the phosphomolybdenum method according to Pierto et al.
Aliquots of 0. Ascorbic acid was used as the reference standard. The radical 1,1-diphenylpicryl-hydrazyl DPPH was used for the determination of free radical-scavenging activity of both extracts [ 19 ]. Fifty microliters of HE or EE diluted in methanol at different concentrations 0. After 30 min of incubation time, the absorbance was measured at nm. The experiment was carried out in triplicate. Then, the dichloromethane was evaporated entirely using nitrogen gas. Also, 40 mL of oxygenated distilled water was added with vigorous shaking.
The absorbance was read at nm. The antioxidant activity AA was calculated regarding inhibition percentage relative to the control using R control and R extract mean rate blanching of control quercetin and extract, respectively.
Briefly, a mixture of g of ground meat and 67 ml of distilled and deionized water with 7. The mixture containing only meat, water, and methanol was used as the control.
BHT 7. Antiglycant activity was in vitro determined using fructose-induced protein glycation models. The method was performed as described by Suzuki et al. Stock solutions of fructose 1. Each solution was sterilized by vacuum filtration using Nalgene cellulose nitrate membrane filters Fisher Scientific Ltd.
The fluorescent intensity was measured at nm excitation and nm emission. The antiglycant activity was expressed as percentage of fluorescent inhibition, using the vehicle as control: F control and F sample mean fluorescence rate of control vehicle and extract or reference standards, respectively. The gels were stained by Coomassie Brilliant Blue R 0. The activity of gelatinases was evidenced as bright regions bleached in the gel. Elastase inhibition activity was determined according to the method of Bieth et al.
The absorbance was read at nm in a microplate. The inhibition rate was measured by the angular coefficient of the curves related to time x substrate concentration. The human embryonic kidney lineage, HEK, was provided by Dr. Marcel Leist University of Konstanz, Germany. The cells were regularly examined regarding mycoplasma contamination and used until 20 passages.
The cells were treated with various extracts concentrations The absorbance was measured at and nm respectively, to DMSO and isopropanol used to dissolve the formazan salt using a microplate reader Thermo Scientific, Waltham, MA, USA and values were calculated in comparison to the control cells.
At least, two experiments in triplicate were performed. Bonferroni test was used for lipid peroxidation and antiglycation activity. The GraphPad Prism 7. Due to their antioxidant capacity, including the ability to chelate metallic ions and to scavenge free radicals, the phenolic compounds, especially flavonoids, are nowadays considered as indispensable components in a variety of nutraceutical, pharmaceutical, medicinal, and cosmetic products, including those destined to prevent the aging phenomenon [ 26 ].
Thus, for better positioning of any herbal drug intended to be used as the active principle of topical pharmaceutical formulations, it is interesting to verify its phenolic and flavonoid contents. Although EE showed the highest total phenolic content, HE exhibited the highest total flavonoid content and higher yield Table 2.
Flavonoids like orientin, iso-orientin, vitexin, isoquercetin, apigenin, and catechin were already identified in C. Although other studies have reported the identification of different flavonoids in C. For this reason, only orientin and iso-orientin were used as chemical markers.
It is well known that the antioxidant activities of plant extracts are commonly related to the presence of phenolic compounds. Also, flavonoids are recognized as tissue protector against reactive oxygen species ROS [ 28 ]. The oxidative stress may lead to harmful cellular modifications, affecting the whole body, causing a variety of disorders, including diabetes, cancer, neurodegenerative diseases, and aging acceleration [ 26 ].
As a part of the natural aging process, endogenous defense mechanisms decrease, while the production of reactive oxygen species increases, resulting in an accelerated skin aging [ 29 ].
The free radical-induced aging theory, as proposed by Harman [ 30 ], suggests that aging and aging-associated degenerative diseases could be attributed to deleterious effects of ROS on various cell components [ 31 ].
Thus, it is intuitive to hypothesize that the topical application of antioxidant compounds may, at least in part, neutralize the skin aging process, and consequently decrease or prevent the external signs of skin aging [ 32 ]. Several methods have been employed for the assessment of antioxidant activities of plant extracts; however, the efficiency cannot be accurately evaluated by a single assay [ 33 ].
For this reason, four different methods were used to evaluate the antioxidant activity of EE and HE. The results showed that both extracts presented a significant antioxidant effect, as described in Table 4. The IC 50 values for EE and HE obtained by DPPH test were the same, and showed a rather high radical-scavenging activity, assuming that EE and HE are crude extracts endowed with several different chemical constituents at minor concentrations, unlike ascorbic acid, which is a pure compound.
The TAC test [ 34 ], performed to evaluate the total antioxidant capacity of plant extracts, also reflected the strength of both extracts as antioxidants. The results clearly demonstrated that both extracts presented a relevant activity.
Those results are quite relevant, as products of lipid peroxidation may react with cell macromolecules to form adducts with significant irreversible effects on cellular functions, which promotes the aging process [ 35 ]. In recent years, the anti-AGE potential of natural or synthetic compounds has gained particular attention by pharmaceutical companies interested in the development of novel antiaging products, as scientific studies have reported the substantial role of AGEs for the aging process [ 36 ].
AGEs are proteins to which a sugar molecule is bound, which can induce widespread tissue and cellular damage [ 37 ], as the original protein functions are inhibited. As skin contains several proteins, including collagen, the formation of these AGEs could be a viable explanation for the skin diminished functioning in aging [ 38 ]. The consequences of AGEs accumulation on the skin are a subject of interest in recent literature aiming at finding the causes for the old aspect of the skin and the decreased skin functions over the age.
As in all organs, long-lived proteins are particularly prone to oxygenation. In the skin, this process affects mainly the dermal extracellular matrix proteins, including collagen, fibronectin, and elastin [ 40 ].
In addition, studies performed in synthetic skin revealed that the glycation process interferes with dermis metabolism, inducing to structural changes in elastic fibers, which reduce skin elasticity and increase MMPs activities [ 41 ]. In this study, the ability of EE and HE to inhibit AGEs formation was evaluated using the BSA-fructose assay, in which the bovine serum albumin is bound to fructose, leading to a glycated protein-sugar complex. The results showed that both EE and HE were capable of preventing the production of the protein-sugar complex.
Cecropia pachystachya , commonly known as Ambay pumpwood ,  is a species of tree in the family Urticaceae. It is native to Argentina, Paraguay and Brazil where it grows near the edges of moist forests. The tree has an open structure, with a small number of branches forking at an obtuse angle, and often forms a parasol-like shape. The twigs are hollow and are filled with mucilage , and both twigs and branches exude a mucilaginous sap when damaged.
Cecropia pachystachya Trécul