GC/MS Profiling, Anti-Collagenase, Anti-Elastase, Anti-Tyrosinase and Anti-Hyaluronidase Activities of a Stenocarpus sinuatus Leaves Extract

Today, skin care products and cosmetic preparations containing natural ingredients are widely preferred by consumers. Therefore, many cosmetic brands are encouraged to offer more natural products to the market, such as plant extracts that can be used for their antiaging, antiwrinkle, and depigmentation properties and other cosmetic purposes. In the current study, the volatile constituents of the hexane-soluble fraction of a Stenocarpus sinuatus (family Proteaceae) leaf methanol extract (SSHF) were analyzed using GC/MS analysis. Moreover, the antiaging activity of SSHF was evaluated through in vitro studies of anti-collagenase, anti-elastase, anti-tyrosinase, and anti-hyaluronidase activities. In addition, an in silico docking study was carried out to identify the interaction mechanisms of the major compounds in SSHF with the active sites of the target enzymes. Furthermore, an in silico toxicity study of the identified compounds in SSHF was performed. It was revealed that vitamin E (α-tocopherol) was the major constituent of SSHF, representing 52.59% of the extract, followed by γ-sitosterol (8.65%), neophytadiene (8.19%), β-tocopherol (6.07%), and others. The in vitro studies showed a significant inhibition by SSHF of collagenase, elastase, tyrosinase, and hyaluronidase, with IC50 values of 60.03, 177.5, 67.5, and 38.8 µg/mL, respectively, comparable to those of the positive controls epigallocatechin gallate (ECGC, for collagenase, elastase, hyaluronidase) and kojic acid (for tyrosinase). Additionally, the molecular docking study revealed good acceptable binding scores of the four major compounds, comparable to those of ECGC and kojic acid. Besides, the SSHF identified phytoconstituents showed no predicted potential toxicity nor skin toxicity, as determined in silico. In conclusion, the antiaging potential of SSHF may be attributed to its high content of vitamin E in addition to the synergetic effect of other volatile constituents. Thus, SSHF could be incorporated in pharmaceutical skin care products and cosmetics after further studies.


Introduction
Skin aging is a complex biological process. It causes various undesirable visible signs such as skin dryness, wrinkles, fine lines, reduction in skin elasticity, and loss of skin strongly inhibited melanogenesis [33]. Additionally, methyl 2,5-dihydroxycinnamate and bis-norstriatol isolated from a methanolic extract of Grevillea robusta leaves was reported to inhibit L-DOPA oxidation by mushroom tyrosinase, with IC 50 values of 69.22 and 65.54 µM, compared to a value of 114.54 µM of a reference standard (kojic acid) [34]. Furthermore, an Oreocallis grandiflora hydroalcoholic extract exhibited photoprotective activity against UV B cell death. It showed a sun protection factor (SPF) of 13.56 at 10 µg/mL, compared to 11.82 and 6.21 of 2-ethylhexyl 4-methoxycinnamate and 2-ethylhexyl 4-(dimethyl amino) benzoate positive controls, respectively, at the same concentration [35].
Despite the great diversity of the reported phytoconstituents and biological activities of many Proteaceous plants, several family members have not been explored yet. These members are expected to be beneficial in the field of medicine and are worth to be investigated. The anti-leishmanial activity of Stenocarpus sinuatus (A. Cunn.) Endl. (Proteaceae) bark extract was previously described [28]. However, the phytoconstituents and biological activities of its leaf extracts have not been investigated yet. S. sinuatus is an Australian tree of medium size and approximately 30 m in height. It grows in tropical and subtropical rainforests. It is sometimes named fire wheel tree or white silky oak due to its ornamental flowers of a bright orange-red color. These flowers produce olfactory cues and ultraviolet markers that attract bees. For example, Pharohylaeus lactiferus, a rare endemic bee, mainly prefers S. sinuatus [36,37]. The interesting previously mentioned antiaging, moisturizing, and skin whitening effects of some Proteaceous plant extracts and oils have encouraged us to investigate S.S. leaf extracts.
The aim of the current study was to investigate the volatile constituents and the biological activities of a Stenocarpus sinuatus extract for the first time. The volatile metabolomic profile of SSHF was characterized by GC/MS. Moreover, the ability of the SSHF extract to inhibit skin aging enzymes (collagenase, tyrosinase, hyaluronidase, and elastase) was evaluated. In addition, in silico docking studies were conducted to demonstrate the mechanism and binding pattern of the four major compounds in SSHF to their targets. Moreover, potential skin toxicity was determined in silico for compounds identified in SSHF.
α-Tocopherol (Figure 2), the major constituent identified herein, is the most biologically active isomer of vitamin E [38]. Vitamin E is composed of naturally occurring lipophilic compounds including α-, β-, γ-, and δ-tocopherols. They differ in the saturation of their side chains and in the methylation degree of their chromanol heads [39]. It was reported in various plants including leaves of Sauropus androgynus (426.8 mg/kg edible part), Capsicum annum red pepper (155.4 mg/kg), Camelia chinensis black tea (183.3 mg/kg), and many others [40].
Many Proteaceous plants were reported to be rich in tocopherol, phytosterols, squalene, and other constituents. For example, Macadamia integrifolia was shown to contain 133.18 mg of vitamin E in 100 g leaves, 2.6% in its kernel, and 5.52% in the pericarp [31]. Macadamia nuts and kernel were reported as rich sources of phytosterols, squalene, and tocopherols [32]. In addition, Gevuina avellana (Chilean hazelnut) displayed a high content of vitamin E [41]. Furthermore, the average total tocopherol content of the nut oil of Gevuina avellana was 1.4 µg/g [42].

In Vitro Assays to Evaluate the Antiaging Potential of Stenocarpus sinuatus Leaf Hexane-Soluble Fraction
The antiaging potential of Stenocarpus sinuatus leaf hexane-soluble fraction was evaluated in vitro. The results revealed a promising activity of SSHF against several enzymes. Different concentrations of the sample were assessed and showed a dose-dependent inhibitory activity against four enzymes ( Figure 3). SSHF displayed considerable antihyaluronidase and anti-collagenase activities, exhibiting IC 50 of 38.8 and 60.03 µg/mL, respectively, approaching that of the standard anti-aging drug epigallocatechin gallate (EGCG) with IC 50 values of 15.5 and 24.7 µg/mL, respectively. In addition, SSHF inhibited tyrosinase, with IC 50 value of 67.5 µg/mL, whereas the kojic acid standard had an IC 50 value of 13.8 µg/mL. Meanwhile, it displayed a lower anti-elastase activity, with IC 50 of 177.5 µg/mL, whereas the IC 50 of the EGCG standard was 18.2 µg/mL.  The identified constituents of SSHF were reported to exert significant biological activities useful in the cosmetic field. Their antiaging, skin whitening, skin moisturizing, and antioxidant effectiveness was observed. These reported beneficial activities encouraged us to evaluate the antiaging potential of the isolated fraction through different in vitro and molecular docking assays.
For example, vitamin E (52.59%) is well known for its antioxidant, free radicle scavenging, and antiaging activities [38]. Moreover, it reduces ultraviolet-induced skin hyperpigmentation. Therefore, its cosmetic application protects the skin from UV damage that aggravates wrinkles, skin dehydration, and loss of elasticity [43]. The depigmentation effect of vitamin E was attributed to its reported anti-tyrosinase and antioxidant activities [44].
Several studies were previously conducted to illustrate the role of vitamin E in the inhibition of the pro-aging enzymes assessed herein. An in vitro study with skin fibroblasts revealed that α-tocopherol reduced the level of expression of collagenase via protein kinase C inhibition. It diminished the transcription of MMP-1 mRNA at 50 µM. This inhibition explained the effect of α-tocopherol against skin age-dependent damage and inflammation [45]. Vitamin E was also used as a reference standard in antioxidant and anti-tyrosinase assays, showing a DPPH scavenging effect with an IC50 of 25.55 µg/mL and inhibition of tyrosinase with a percentage of inhibition of 36.27 ± 3.73% [46].
An in vivo study on 10 female volunteers examined the effectiveness of a vitamin E microsphere formulation. Enhancement of skin elasticity and skin moisture and decrease of wrinkles volume were reported [47]. Consequently, vitamin E can be used either alone or in combination with other ingredients in cosmetic preparations to improve skin integrity and pigment appearance besides reducing skin aging [43]. Thus, the high content of vitamin E in SSHF suggests a significant antiaging activity of this extract.
Phytosterol constituents identified in SSHF such as γ-sitosterol (8.65%) and Campesterol (0.66%) were reported to be biologically active, exhibiting emollient and antiaging effects and protecting the skin against UV damage. Moreover, the ability of phytosterols to inhibit MMP-1, constrain collagen degradation, and enhance collagen synthesis was experimentally demonstrated in human keratinocytes [48]. Besides, they were reported to stimulate the synthesis of hyaluronic acid, increase the thickness of the epidermis, enhance skin elasticity, and reduce skin roughness. Therefore, they can be incorporated in antiaging creams as well as sun-care products [49].

In Vitro Assays to Evaluate the Antiaging Potential of Stenocarpus sinuatus Leaf Hexane-Soluble Fraction
The antiaging potential of Stenocarpus sinuatus leaf hexane-soluble fraction was evaluated in vitro. The results revealed a promising activity of SSHF against several enzymes. Different concentrations of the sample were assessed and showed a dosedependent inhibitory activity against four enzymes ( Figure 3). SSHF displayed considerable anti-hyaluronidase and anti-collagenase activities, exhibiting IC50 of 38.8 and 60.03 µg/mL, respectively, approaching that of the standard anti-aging drug epigallocatechin gallate (EGCG) with IC50 values of 15.5 and 24.7 µg/mL, respectively. In addition, SSHF inhibited tyrosinase, with IC50 value of 67.5 µg/mL, whereas the kojic acid standard had an IC50 value of 13.8 µg/mL. Meanwhile, it displayed a lower anti-elastase activity, with IC50 of 177.5 µg/mL, whereas the IC50 of the EGCG standard was 18.2 µg/mL.
The identified constituents of SSHF were reported to exert significant biological activities useful in the cosmetic field. Their antiaging, skin whitening, skin moisturizing, and antioxidant effectiveness was observed. These reported beneficial activities In addition, many diterpenes isolated from plants have been reported to show potential antioxidant and anti-inflammatory activities [50,51]. Phytol diterpene identified in SSHF (4.04%) was used in cosmetics to inhibit cellular senescence, in particular of keratinocytes, induced by oxidative stress [52]. In It also exhibited antioxidant, anti-inflammatory, antimicrobial, cytotoxic, and immune-modulating activities [53]. Previous molecular docking studies revealed that phytol can bind to amino acid residues in the catalytic domain of MMP-1 (collagenase enzyme), with binding energy (−7.06 kcal/mol) comparable to that (−8.05 kcal/mol) of the reference compound doxycycline [54].

In Silico Docking Studies on the Target Enzymes
Driven by the promising inhibitory activity of the extract against collagenase, elastase, hyaluronidase, and tyrosinase, an in silico docking study was conducted. Docking studies are performed to identify the possible binding mechanism and pattern of compounds to potential targets [58,59]. The main compounds that we identified were previously reported to exert antiaging activity in several studies. For example, vitamin E was found to inhibit tyrosinase and collagenase in vitro [45,46]. In addition, neophytadiene showed potent binding to tyrosinase in a previous in silico study [56]. Furthermore, phytosterols were reported to enhance the synthesis of collagen and hyaluronic acid [48,49]. Therefore, we assumed that the major identified compounds might be responsible for the observed activities of SSHF. In this respect, the X-ray structures of the four enzymes were downloaded from the PDB to conduct the docking assays. First, the applied docking parameters were validated by re-docking each ocrystalized ligand into its corresponding active site. The calculated RMSD values between Therefore, in the current study, the observed significant activities of SSHF can be explained by the antiaging, skin whitening, and antioxidant effects previously reported for its identified components. Thus, it can be concluded that the significant inhibitory activity of SSHF to hyaluronidase collagenase, tyrosinase, and elastase may be attributed to its high content of vitamin E and the synergetic effects of all its constituents.

In Silico Docking Studies on the Target Enzymes
Driven by the promising inhibitory activity of the extract against collagenase, elastase, hyaluronidase, and tyrosinase, an in silico docking study was conducted. Docking studies are performed to identify the possible binding mechanism and pattern of compounds to potential targets [58,59]. The main compounds that we identified were previously reported to exert antiaging activity in several studies. For example, vitamin E was found to inhibit tyrosinase and collagenase in vitro [45,46]. In addition, neophytadiene showed potent binding to tyrosinase in a previous in silico study [56]. Furthermore, phytosterols were reported to enhance the synthesis of collagen and hyaluronic acid [48,49]. Therefore, we assumed that the major identified compounds might be responsible for the observed activities of SSHF. In this respect, the X-ray structures of the four enzymes were downloaded from the PDB to conduct the docking assays. First, the applied docking parameters were validated by re-docking each o-crystalized ligand into its corresponding active site. The calculated RMSD values between the docked pose and the co-crystalized pose were 0.77, 1.2, 0.61, and 0.81 Å for collagenase, elastase, hyaluronidase, and tyrosinase, respectively, indicating that the docking protocol was valid. The docking of EGCG to hyaluronidase, collagenase, and elastase resulted in docking scores of −8.19, −8.8, and−8.9 Kcal/mole, while the docking of kojic acid to its target enzyme resulted in a docking score of −4.62 Kcal/mole. The docking of the four major compounds to the four enzymes resulted in good acceptable scores, comparable to those of the reference compounds EGCG and kojic acid. Table 2 summarizes the docking interactions of the four compounds with the four potential target enzymes and the corresponding docking scores. Taking into account the hydrophobic nature of the isolated compounds composed mainly of a hydro-carbonic skeleton, most of the observed interactions with the four enzymes were found to be hydrophobic in nature (see Figures 4-7). In conclusion, from the major extract, the four identified compounds had the ability to strongly interact with the four enzymes collagenase, elastase, hyaluronidase, and tyrosinase, achieving acceptable docking scores that sometimes exceeded those of the reference compounds. These acceptable scores were achieved through the establishment of many hydrophobic interactions. Thus, the observed strong binding interactions validated the activities of SSHF and suggest possible mechanisms of action.

In Silico Toxicity Study
The SSHF constituents showed no toxicity in silico using Pro-toxll for the prediction of any potential toxicity. In addition, no potential skin toxicity could be predicted when using Pred-Skin 3 and a Bayesian model. The results are shown in Table 3. Furthermore, no reported harmful or toxic effects could be established for any of the compounds identified in SSHF.

In Silico Toxicity Study
The SSHF constituents showed no toxicity in silico using Pro-toxll for the prediction of any potential toxicity. In addition, no potential skin toxicity could be predicted when using Pred-Skin 3 and a Bayesian model. The results are shown in Table 3. Furthermore, no reported harmful or toxic effects could be established for any of the compounds identified in SSHF.

In Silico Toxicity Study
The SSHF constituents showed no toxicity in silico using Pro-toxll for the prediction of any potential toxicity. In addition, no potential skin toxicity could be predicted when using Pred-Skin 3 and a Bayesian model. The results are shown in Table 3. Furthermore, no reported harmful or toxic effects could be established for any of the compounds identified in SSHF.

Plant Material
The leaves of Stenocarpus sinuatus were collected in Spring 2020 from El-Abd farm, Cairo Alexandria desert road, Egypt. Plant material was kindly identified and authenticated by Mrs. Treiz Labib, Plant Taxonomy Consultant at the Ministry of Agriculture, Egypt. A voucher specimen (PHG-P-SS-326) was deposited in the Herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University.

Preparation of the Extracts
The air-dried leaves were ground and extracted with methanol (12 L × 3). The extract was filtered, and the solvent was evaporated using a rotary evaporator under reduced pressure and lyophilized to yield 350 g of methanol extract. The extract was then defatted with hexane (3 L × 3). The hexane soluble fraction was evaporated under reduced pressure to yield 45 g of residue. The hexane soluble fraction was kept in a tightly closed container for further analysis.

GC/MS Analysis of the Hexane-Soluble Fraction
GC/MS analysis of the hexane-soluble fraction was carried out using a Shimadzu QP2010 gas chromatograph coupled to a quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan) [60][61][62] and equipped with an Rtx-5MS fused bonded capillary column (Restek, PA, USA, dimensions, 30 m × 0.25 mm i.d. × 0.25 µm film thickness) and a split-splitless injector. Helium was used as a carrier gas at a flow rate of 1.37 mL/min, and an injection volume 1 µL of the diluted sample (1% v/v) was employed in split mode at a split ratio of 1:15. The injector temperature was adjusted at 280 • C, the oven temperature was kept at 50 • C for 3 min, then programmed to reach 300 • C at a rate of 5 • C/min and kept constant at 300 • C for 5 min. As the MS operating parameters, the ion source temperature was maintained at 220 • C, and the separation was carried out at 70 eV in electron ionization (EI) mode, filament emission current 60 mA, and scanning from 35 to 500 amu. The compounds were identified by the comparison of their retention indices (RI) and mass spectra (MS) to values reported in the NIST mass spectral library database (similarity index > 90%) and in the literature [63][64][65][66][67][68][69][70][71][72]. The retention indices were calculated relative to those of a homologous series of standard n-alkanes (C8-C28) injected under the same conditions. The results was processed by GCMSsolution Workstation Software for Gas Chromatography-Mass Spectrometry.

Anti-Collagenase Assay
The anti-collagenase assay was performed spectrophotometrically according to the method reported by Thring et al. [73] with minor modifications. The assay was carried out in 50 mM Tricine buffer (pH 7.5 with 10 mM CaCl 2 and 400 mM NaCl). Collagenase produced by the bacterium Clostridium histolyticum (ChC-EC.3.4.23.3) was first dissolved in the buffer to an initial concentration of 0.8 unit/mL based on the supplier's activity data. N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) was used as a synthetic substrate, dissolved in Tricine buffer to a concentration of 2 mM. The samples in a concentration range of 1000-7.81 µg/mL were incubated at room temperature with the prepared collagenase in Tricine buffer for 15 min; then, the synthetic substrate was added to the samples to start the reaction. The absorbance values were measured at 490 nm using a microplate reader (TECAN, Inc., Durham, NC, USA). A positive control (EGCG) was used, while the negative control consisted of water. The percentage of collagenase inhibition was calculated according to (%) = [1 − (S/C) × 100], where "S" is the corrected absorbance of the controls (in the absence of a sample). The inhibitory concentration 50, inhibiting 50% of the enzyme (IC50), was estimated from the graph plots of the dose-response curve for each sample concentration by GraphPad Prism software (San Diego, CA, USA).

Anti-Elastase Assay
The anti-elastase assay was performed spectrophotometrically as previously reported by Kim et al. [74] with minor modifications. Elastase from pancreatic porcine was dissolved in sterile water to obtain a stock solution at a concentration of 3.33 mg/mL. N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN) was used as a substrate, dissolved in Tris-HCL buffer (pH 8) at 1.6 mM. The samples in the concentration range of 1000-7.81 µg/mL were incubated at room temperature with the prepared elastase solution in the buffer for 15 min; then the synthetic substrate was added to the samples to start the reaction. The final mixture with a total volume 250 µL contained buffer, substrate (AAAPVN) 0.8 mM, 25 µg of test extract, and 1 µg/mL of PE. A positive control (EGCG) was used, while the negative control consisted of water. The absorbance was measured at 400 nm in a 96-well microtiter plate using a microplate reader (TECAN, Inc., Durham, NC, USA). The percentage of elastase inhibition was calculated according to (%) = [1 − (S/C) × 100], where "S" is the corrected absorbance of the tested samples, while "C" is the corrected absorbance of the controls (in the absence of a sample). The inhibitory concentration 50 (IC50) was estimated from the graph plots of the dose-response curves at each sample concentration by GraphPad Prism software (San Diego, CA, USA).

Anti-Tyrosinase Assay
This assay was performed spectrophotometrically as previously described in the literature [75]. L-DOPA was used as a substrate. The reaction mixture in a total volume of 1000 µL contained 15 µL of mushroom tyrosinase (2500 U/mL), 685 µL of phosphate buffer (pH 6.5, 0.05 M), 100 µL of 5 mM L-DOPA, and 200 µL of the samples in the concentration range of 1000-7.81 µg/mL. The positive control kojic acid was used, whereas the negative control consisted of water. After the addition of the substrate (L-DOPA), the absorbance was measured at 475 nm using a microplate reader (TECAN, Inc., Durham, NC, USA); each measurement was carried out in triplicate. The percentage of tyrosinase inhibition was calculated according to (%) = [1 − (S/C) × 100], where "S" is the corrected absorbance of the tested sample, while "C" is the corrected absorbance of the controls (in the absence of a sample). The inhibitory concentration 50 (IC50) was estimated from the graph plots of the dose-response curves at each sample concentration by GraphPad Prism software (San Diego, CA, USA). IC50 is the concentration of the sample needed to inhibit 50% of tyrosinase activity under the used assay conditions.

Anti-Hyaluronidase Assay
The hyaluronidase inhibitory assay was performed following the fluorimetric Morgan-Elson method reported by Reissig et al. [76] and modified by Takahashi et al. [59]. The reaction mixture in 2 mL test tubes contained 25 µL of calcium chloride (12.5 mM), 100 µL of hyaluronic acid substrate (1 mg/mL in 0.1 M acetate buffer; pH 3.5), 12.5 µL each of hyaluronidase (1.5 mg/mL), and the sample (2.8 mg/mL). The range of sample concentration for the hyaluronidase inhibition assay was 1000-7.81 µg/mL. Twenty-five microliters of KBO 2 (0.8 M) was added to tubes, which were then placed in a water bath at 100 • C for 3 min, followed by cooling at room temperature. Then, 800 µL of DMAB (4 g DMAB in 5 mL 10 N HCl and 40 mL acetic acid) was added. Then, the tubes were incubated for 20 min at room temperature, and their contents were transferred to wells in a 96-well plate. Fluores-cence was detected by a Tecan Infinite microplate reader (TECAN, Inc., Durham, NC, USA) at 545 nm excitation wavelength and 612 nm emission wavelength. The inhibitory concentration 50 (IC50), the concentration required to inhibit 50% of hyaluronidase activity under the used assay conditions, was estimated from the graph plot of the dose-response curves at each concentration using GraphPad Prism software (San Diego, CA, USA).
3.6. In Silico Studies 3.6.1. In Silico Docking Studies on the Target Enzymes The docking studies in the current work were performed using the platform of Vina autodock and M.G.L. tools 1.5.6 [77,78]. The crystal structures of the four potential targets, namely, collagenase, elastase, hyaluronidase, and tyrosinase were downloaded from the protein data bank using the following PDB IDs: 456c, 6qeo, 1fcv, and 5m8q, respectively. The four major compounds, vitamin E (α-tocopherol), γ-sitosterol, neophytadiene, and β-tocopherol, in addition to EGCG and kojic acid, were sketched using MOE builder and then energy-minimized under Amber12: EHT force field using a steepest descent algorithm [79,80]. The lowest energy conformations of each of the six compounds as well as of the four targets were saved in pdbqt format as a prerequisite for the Vina autodock. The applied docking approach was firstly validated by re-docking each co-crystalized ligand into the active site of its corresponding enzyme. This step was followed by RMSD calculation between the co-crystalized and the docked poses for each enzyme [81]. Finally, the four major compounds were docked in the binding site of each enzyme using the validated docking protocol. Besides, EGCG was docked in hyaluronidase, collagenase, and elastase, while kojic acid was docked in tyrosinase. Biovia Discovery Studio visualizer was implemented in docking analysis and used to generate the binding interaction images.

In Silico Toxicity Study
In addition, an in silico toxicity study of the identified compounds was carried out. Any potential toxicities were predicted by Pro-toxII, while potential skin toxicity was predicted by Pred-Skin 3 using a Bayesian model [82,83].

Statistical Analysis
The performed assays were carried out in triplicates, and the values are expressed as mean ± SD. For the determination of the in vitro anti-collagenase, anti-tyrosinase, antielastase, anti-hyaluronidase activities, the (IC50) was estimated from the graph plots of the dose-response curves at each sample concentration by Graph Pad Prism software (San Diego, CA, USA). The IC50 is the concentration of the sample needed to inhibit 50% of the tested enzyme activity under the used assay conditions.

Conclusions
The present study investigated the volatile phytoconstituents and the biological activities of SSHF for the first time. The GC/MS analysis revealed that α-tocopherol, γ-sitosterol, neophytadiene, and β-tocopherol are the major compounds of the lipophilic fraction. In addition, SSHF showed significant anti-hyaluronidase, anti-collagenase, anti-tyrosinase activity, and low anti-elastase activity. The four major compounds achieved acceptable docking scores in the active sites of the target enzymes. The docking scores sometimes exceeded those of the reference compounds. In addition, the constituents of SSHF showed no skin toxicity in an in silico study. Based on these studies and previous reports, it can be concluded that the antiaging activities of SSHF may be attributed to its high content of vitamin E as well as to the synergistic action of all its constituents. Therefore, SSHF could be considered an excellent antiaging candidate that could be incorporated in pharmaceutical skin care products and cosmetics after further clinical trials.

Conflicts of Interest:
The authors declare there is no conflict of interest.