**4. Discussion**

Practitioners of traditional and folk medicine have used the placenta to treat a wide range of ailments. Along with its medicinal uses, it has also been used as an anesthetic in

various clinical settings, including internal medicine, general surgery, ENT, ophthalmology, orthopedic surgery, plastic surgery, dermatology, obstetrics, and gynecology [9]. The main amino acids in SPE are glutamic acid, aspartic acid, lysine, and leucine. Eight essential amino acids constitute 31.20 g/100 g of SPE or 39.48 percent of total amino acids. Additionally, several cytokines and growth factors present in placenta extract contribute to their pharmacological activities [23].

Reactive oxygen species (ROS), which cause oxidative damage and lipid peroxidative injury, are produced throughout the aging process [24]. Free radicals and their metabolic byproducts must be removed by enzymes of antioxidant systems. The key enzymes in antioxidant systems are SOD and catalase [25]. Superoxide anion free radicals and hydrogen ions are dismutated into H2O2 and O2 by the antioxidant enzyme SOD, which is crucial for cleaning up free radicals [26]. Catalase eliminates oxidative risks by accelerating the synthesis of H2O and O2 from H2O2. One of the byproducts of lipid peroxidation is TBARS. During the aging process, TBARS levels increase [27]. In this study, we used these three defining indices to assess the antioxidant mechanism of SPE. We found that in SAMP8 mice, SOD and catalase activities decreased and the level of TBARS increased; however, SPE treatment for 13 weeks increased SOD and catalase antioxidant enzyme activities and lowered TABRAS levels in brain tissue. Antiaging processes may include regulating the activity of these antioxidant enzymes. There is strong evidence to support the original free radical hypothesis of aging, which states that free radical damage caused by numerous endogenous ROS is related to aging but is most likely the cause of it. Uracil, tyrosine, phenylalanine, and tryptophan are the main antioxidant substances found in placenta extract. These elements are responsible for 59% of PE's anti-oxidative actions. Freeze-dried placenta powder delays D-galactose-induced aging in female KM mice by improving immunity and reducing LPO in the brain [28], which is in line with our present findings. Preparations from deer fetuses and placenta produce an antiaging effect by reducing monoamine oxidase activities in the brain and liver and lipofuscin activities in the heart and brain of aging rats [29].

High doses (1000 mg/kg) of swine placenta extract powder administered for 28 days do not produce any adverse effects in rats. Male and female rats treated with swine placenta powder showed no changes in hematological parameters and no changes to vital organs and tissues including the kidneys, heart, lung, liver, spleen, brain, adrenals, epididymides, prostate, seminal vesicles, thymus, testes, or ovaries. Uterus histology and organ weights were also unaffected [30]. In our study, we checked both male and female SAMP8 mice every day for symptoms of toxicity and death. In addition, we periodically checked the body weight, food and water consumption, and physical and visual health of the animals. We also carried out regular urinalysis and performed clinical biochemistry assessments of blood and plasma samples. We found that a higher dose of SPE (184.5 mg/kg BW) did not induce any changes in serum biochemical parameters or any histological changes in vital organs. This is in line with the previous findings and suggests that SPE is safe for human consumption.

Manufacturers employ a variety of techniques to produce placental extracts using raw materials from human, bovine, and porcine placentas. As a result, the components of placental extracts can be quite complex and may widely vary between products. Placental extracts include N-acetylneuraminic acid, glucosamine, omega-3 fatty acids, various fatty acids, and many other amino acids and nucleotides that have antioxidative or antiinflammatory and wound-healing activities [31–33]. We postulated that most of these compounds/molecules would also be present in our sheep placenta extract. However, we were unable to validate the crucial elements and signaling pathways important for the prevention of aging. More research is needed to understand the molecular mechanism underpinning the antiaging activity of SPE, and this may be a challenging task.

As well as the aging index, serum biochemical parameters might be used as an indicator of aging. The liver function markers, ALT and AST, and the kidney function markers, creatinine and uric acid, are key indicators of tissue damage during the aging process [34]. However, in our study, these biochemical parameters did not significantly change after SPE administration. Inflammation plays a key role in the process of senescence [35]. Levels of cytokine IL-6 increase in elderly individuals, in comparison with young people, but levels of IL-1β and TNF-α do not increase [36]. In our study, we found no significant changes in levels of IL-6 or TNF-α.

In this study, we investigated the effect of different doses of SPE for 13 weeks on male and female SAMP8 in terms of the aging index, serum biochemical levels, and antioxidant status. We found that medium and high doses of sheep placenta reduced the appearance changes in aging, lowered levels of brain lipid peroxides, and increased SOD and catalase activities, thereby enhancing the antioxidant capacity of the body and helping to reduce the damage of oxidative stress, with the overall effect of delaying the aging process. SPE is rich in peptides and amino acids, whose strong antioxidant activities might contribute to antiaging effects. However, further characterization and molecular studies of SPE are needed to better understand its antiaging effects. In conclusion, SPE supplementation reduces the aging index and lowers the oxidative stress caused by the aging process in male and female mice, without toxicity, suggesting the value of SPE as an effective antiaging treatment.

**Author Contributions:** Conceptualization, W.-T.L.; methodology, C.-P.O.Y. and M.-Y.C.; validation, W.-C.L. and Y.-M.Y.; formal analysis, W.-C.L.; investigation, M.-Y.C. and Y.-J.H.; resources, M.-F.W.; data curation, M.-F.W.; writing—original draft preparation, M.-F.W. and W.-T.L.; writing—review and editing, M.-F.W. and W.-T.L.; supervision, W.-T.L.; project administration, M.-F.W.; funding acquisition, W.-T.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Providence University, fund number: 109: PU109-11150-A044, and Tunghai University Fund number: THU 111611.

**Institutional Review Board Statement:** All the animal treatment procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, raised 1996). Animal experiment procedures in the present study were approved by the Institutional Animal Care and Use Committee (IACUC) of Providence University (Approval No.: 20201218 A008).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

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

#### **References**

