3.4. In Vitro Assays

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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To validate the previously discussed in silico findings, we tested compounds 2, 3, and 5  for their inhibitory activity against hyaluronidase, xanthine oxidase, and tyrosinase enzymes in vitro. As presented in Table 3, compound 3 was identified as a potent hyaluronidase inhibitor followed by compound 2 with IC50 values of 9.5 ± 0.48 and 13.7 ± 1.08 µM,  respectively. The known 6-O-palmitoyl-L-ascorbic acid (IC50 2.033 ± 0.1 µM) was used as a positive control. Compound 5 was inactive against hyaluronidase. Regarding xanthine oxidase, compound 3 was significantly able to inhibit its activity with the IC50 value of 6.39 ± 0.36 µM, while both compounds 2 and 5 showed weak or inactivity, the known L-mimosine (IC50 3.63 ± 0.18 µM) was used as a positive control. Finally, compound 5  was the most active compound against tyrosinase, with an IC50 value of 8.67 ± 0.44 µM,  while compounds 2 and 3 were inactive or weakly active, and the known kojic acid (IC50 6.52 ± 0.33 µM) was used as a positive control. These in vitro results revealed the potential of C. reticulata seed-derived flavonoids, particularly compounds 2, 3, and 5, as healthy skin-promoting agents via their inhibition of the activity of several relevant enzymes (i.e.,  hyaluronidase, xanthine oxidase, and tyrosinase enzymes). Additionally, they showed the applicability of using different in silico-based analyses as a preliminary screening step in the characterization of biological activity in natural products.

Extracellular matrix (ECM) degradation is the primary cause of skin aging [32]. Collagenase and gelatinases (MMP-2) are matrix metalloproteinases (MMPs) that have a role in ECM degradation [33]. As a result, the skin's tensile strength is depleted. Roughness,  wrinkling, and dehydration of the skin still occur often, as do various pigment anomalies such as hy-po-/hyper-pigmentation [32,34]. Tyrosinase inhibitors have been studied for the treatment of hyperpigmentation of the skin.

The enzyme tyrosinase converts tyrosine to melanin [35]. As a result, tyrosine inhibitors play an important role as skin-lightening agents [36]. Hyaluronic acid (HA)  production is being studied for the treatment of skin wrinkles. The presence of wrinkles and skin moisture have both been linked to HA. HA also deals with tissue improvement,  including immune system response augmentation through inflammatory cell activation and fibroblast injury [37,38]. Hyaluronidase is a proteolytic enzyme found in the dermis that is responsible for the breakdown of hyaluronan in the extracellular matrix, resulting in visible signs of skin aging [39]. 

As a result, hyaluronidase inhibitors are crucial in the treatment of skin wrinkles. XO  is also a major source of oxidants and plays a role in several oxidative stress-related diseases. Because of the ongoing oxidative stress situation, aging is associated with the progressive deregulation of homeostasis [40]. As a result, XO inhibitors affect skin aging treatment.

The findings of this study showed that C. reticulata seed-derived flavonoids, particularly compounds 2, 3, and 5, can promote healthy skin by inhibiting the activity of hyaluronidase, xanthine oxidase, and tyrosinase enzymes. Compound 3 was found to be a potent hyaluronidase inhibitor, followed by compound 2 with IC50 values of 9.5 0.48 and 13.7 1.08 M, respectively. With an IC50 value of 6.39 0.36 M, compound 3 was able to strongly inhibit the activity of xanthine oxidase. With an IC50 value of 8.67 0.44 M,  compound 5 was the most potent chemical against tyrosinase (Table 3).

The societal, therapeutic, and commercial difficulties posed by non-healing wounds are growing as our society ages. As a result, studying the impact of aging on wound healing has become a popular issue [41]. Skin functions deteriorate with age due to anatomical and morphological changes directed by innate factors such as historical makeup, changes in hormone stages, and exogenous factors such as sun exposure and cigarette smoking [42]. Aging skin changes not only affect wound healing but also make the skin particularly susceptible to wounds. Devaluation of nerve endings, for example, diminishes pain sensitivity, increasing the risk of damage, and epidermal degeneration causes the skin to become more susceptible to mechanical forces.

The growth of chronic wounds is aided by immunosenescence. Microvascular disruptions may also reveal the fate of ischemic lesions [41,42].

Flavonoids are found in abundance as bioactive secondary metabolites. They are found in a variety of medicinal plants that are used to improve wound healing [43]. The topical application of kaempferol 1, which has anti-inflammatory and antioxidant properties,  was found to have healing effects on incisional and excisional wounds in diabetic and non-diabetic rats [44]. Kaempferol 1 mediated these effects by increasing wound collagen and hydroxyproline output, improving wound protection, speeding wound closure, and hastening re-epithelialization.

Furthermore, kaempferol and its glycosides derivatives 2–3 exhibited astringent and antimicrobial properties that were found to be useful for wound shrinkage and enhancing the rate of epithelialization in male Wistar rats using an excision and incision wound model,  as well as encouraging the movement of CCD-1064sk fibroblasts into a scratch wound assay on Ha-CaT keratinocytes [45,46]. Moreover, isoflavonoid (e.g., 2-hydroxy genistein, 4) has been shown to promote wound healing by increasing tensile strength, reducing inflammation, and inhibiting collagenase, hyaluronidase, and elastase enzymes [47].

Genistein, a 2-deoxy derivative of 2-hydroxy genistein 4, has been linked to soy's beneficial effects, particularly in the context of aging. Cut intrinsic estrogen leads to a  range of age-related diseases in postmenopausal women, including protracted cutaneous wound healing. Genistein accelerated wound healing while suppressing the inflammatory response. Genistein’s actions were limited to interfering with estrogen receptor-dependent signaling [48]. In sham OVX rats, genistein reduced tissue transglutaminase-2, TGF-1, and vascular endothelial growth factor, indicating that genistein derivatives have anti-aging aesthetic characteristics [49].

Hesperidin has sufficient healing benefits on injured skin. Hesperidin can thus be utilized as a supplement or alternative to other wound-healing agents [50–52]. Aside from flavonoids, fatty acid esters of glycerol [53,54], acrylic acid derivatives [55], and sterols [56]  all had similar wound-healing effects. The potential of C. reticulata seed extract in age-related characteristics of cutaneous wound healing was disclosed in this literature, however, more in vivo testing is needed.

4. Conclusions

Herein, we investigated the chemical composition of C. reticulata seeds via stepwise chromatographic isolation and the subsequent spectroscopic-based structural identification. Flavonols were found to be the most prevalent type of flavonoids in the investigated seeds instead of the well-known predominance of flavanones and flavones in the aerial parts, including the fruits. Additionally, several other common oligosaccharides, sterols, and fatty acids were also found to be major metabolites. In a silico-based study of the isolated flavonoids aiming at characterizing their pharmacological effects highlighted their potential as hyaluronidase, xanthine oxidase, and tyrosinase inhibitors. Further MDSbased investigation selected compounds 2, 3, and 5 to be the most promising candidates against these skin-related enzymes. Final in vitro enzyme assays revealed the potential of these compounds (i.e., 2, 3, and 5) as skin-promoting agents via their inhibitory activity against hyaluronidase, xanthine oxidase, and tyrosinase activity. This study highlighted the waste product of C. reticulata seeds as a very good source of healthy skin-promoting phytochemicals and age-linked features of cutaneous wound healing. Additionally, it revealed the power of integrating inverse docking with MDS experiments in characterizing the biological activities of natural products.

Supplementary Materials: The following supporting information can be downloaded at: https://  www.mdpi.com/article/10.3390/antiox11050984/s1, Figure S1: 1H NMR spectrum of compound 1  measured in CD3OD-d4 at 400 MHz; Figure S2: DEPT-Q NMR spectrum of compound 1 measured in CD3OD-d4 at 100 MHz; Figure S3: 1H NMR spectrum of compound 2 measured in CD3OD-d4 at 400 MHz; Figure S4: DEPT-Q NMR spectrum of compound 2 measured in CD3OD-d4 at 100 MHz; Figure S5: 1H NMR spectrum of compound 3 measured in CD3OD-d4 at 400 MHz; Figure S6: DEPT-Q NMR spectrum of compound 3 measured in CD3OD-d4 at 100 MHz.; Figure S7: 1H NMR spectrum of  compound 4 measured in CD3OD-d4 at 400 MHz; Figure S8: DEPT-Q NMR spectrum of compound 4 measured in CD3OD-d4 at 100 MHz; Figure S9: 1H NMR spectrum of compound 5 measured in DMSO-d6 at 400 MHz; Figure S10: DEPT-Q NMR spectrum of compound 5 measured in DMSO-d6  at 100 MHz: Figure S11: 1H NMR spectrum of compound 6 measured in CD3OD-d4 at 400 MHz; Figure S12: DEPT-Q NMR spectrum of compound 6 measured in CD3OD-d4 at 100 MHz; Figure S13: HSQC spectrum of compound 6 measured in CD3OD-d4; Figure S14: HMBC spectrum of compound 6 measured in CD3OD-d4; Figure S15: 1H NMR spectrum of compound 7 measured in CD3OD-d4 at 400 MHz; Figure S16: DEPT-Q NMR spectrum of compound 7 measured in CD3OD-d4 at 100 MHz; Figure S17: H NMR spectrum of compound 8 measured in DMSO-d6 400 MHz; Figure S18: DEPT-Q NMR spectrum of compound 8 measured in DMSO-d6 at 100 MHz; Figure S19: 1H NMR spectrum of  compound 9 measured in DMSO-d6 at 400 MHz; Figure S20: DEPT-Q NMR spectrum of compound 9  measured in DMSO-d6 at 100 MHz; Figure S21: 1H NMR spectrum of compound 10 measured in DMSO-d6 at 400 MHz; Figure S22: DEPT-Q NMR spectrum of compound 10 measured in DMSO-d6  at 100 MHz; Figure S23: 1H NMR spectrum of compound 11 measured in DMSO-d6 at 400 MHz; Figure S24: DEPT-Q NMR spectrum of compound 11 measured in DMSO-d6 at 100 MHz; Figure S25: 1H NMR spectrum of compound 12 measured in CDCL3-d at 400 MHz; Figure S26: DEPT-Q NMR  spectrum of compound 12 measured in CDCL3-d at 100 MHz; Figure S27: 1H NMR spectrum of  compound 13 measured in CDCL3-d at 400 MHz; Figure S28: DEPT-Q NMR spectrum of compound 13 measured in CDCL3-d at 100 MHz.

Author Contributions: Conceptualization: U.R.A., A.H.E., and A.M.S., methodology: A.H.E., A.M.S., T.A.-W., S.S., and M.M.A.-S.; software: A.H.E., M.A., E.M.M., and S.I.; formal analysis: M.M.G., A.M.S., and A.H.E.; investigation: U.R.A., A.H.E., and T.A.-W.; resources: S.S., M.M.A.-S., M.A., E.M.M., and S.I.; data curation: U.R.A., A.H.E., and A.M.S.; writing—original draft: U.R.A., A.H.E., and A.M.S.;  writing—review and editing: U.R.A., A.H.E.; project administration: T.A.-W. and S.S.; funding acquisition: M.M.A.-S., M.A., and E.M.M. All authors have read and agreed to the published version of the manuscript.

Funding: Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R25), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Acknowledgments: The authors deeply acknowledge Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R25), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors deeply acknowledge the researcher support program (TUMA-Project-2021-6) of AlMaarefa University, Riyadh, Saudi Arabia for supporting steps of this work.

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

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