Introduction

The aging process is defined as a gradual, unavoidable loss of several body functions1. As age progresses, the brain undergoes biological, psychosocial, neuroanatomical, and neurophysiological changes that are all associated with a decline in cognitive function2. There is accumulating evidence that increasing male age has a considerable detrimental influence on spermatogenesis and fertilization3. The spermatogenesis microenvironment reveals major anomalies with age, such as reduced number, organelle aging, aberrant hormone production, and deficiencies of the blood–testicular barrier4. Aging is characterized by a gradual decrease in tissue and organ function. According to the oxidative stress theory of aging, the accumulation of harmful reactive oxygen and nitrogen free radicals leads to age-related functional impairments. In addition, oxidative stress contributes to a number of age-related illnesses, such as sarcopenia and frailty (., cardiovascular diseases, obstructive pulmonary disease, diabetes, renal disease, neurodegenerative diseases, and cancer). Antioxidant defenses mitigate the detrimental effects of free radicals, which are produced by a range of internal and environmental processes. Oxidative stress is brought on by an imbalance between the production of free radicals and antioxidant defenses5. Phytochemical prevention of serious health conditions has gained global recognition, with studies on the anti-aging properties of plant extracts garnering substantial attention.

(MO) is a medicinal plant belonging to the family Moringaceae. The MOS has recently drawn a lot of interest because of its nutritional value and health advantages6. MOS (fresh, powdered, or cooked) includes a wide range of nutrients, such as significant sulfur amino acids, protein, and minerals7. Recently, bioactive peptides generated from natural plants have gained attention in the health and pharmaceutical industries8. According to Jain .9, MOS contains around 52% of all necessary amino acids, making it a possible source of functional protein isolate. Because of the harmonized amino acid content, MOS protein could be utilized instead of other sources of protein for human diets. MOS protein contains significant levels of cysteine and methionine (43.6 g/kg protein), which are comparable to chicken eggs, cow, and human milk10. MOS has displayed a plethora of bioactivities such as antioxidant11, anti-inflammatory12, antimicrobial13, antiviral14, liver-protective15, anti-diabetic16, antitumor17, and cardio-protective activities18.

Previous research has concentrated on the utilization of MOS and its protective mechanisms in various tissues rather than the brain. As a result, the current study employs MOS to assess its effect as an antioxidant against oxidative stress produced by aging, such as serum biochemical disturbance and brain and testicular tissue damage.

Methods

All materials were of analytical grade and were purchased from commercial stores.

Statistical Analysis

Results are represented as mean values ± standard error. A parametric test was used to determine whether the data had a normal distribution, and thereafter, various assessments were statistically analyzed using one-way ANOVA tests, followed by Tukey's HSD multiple comparisons as a post-hoc test, to identify the significant differences between the various groups. A p-value of < 0.05 was considered statistically significant. The software SPSS statistical version 20 (SPSS® Inc., USA) was used for all statistical evaluations.

MOS (1g/15 ml)
Compounds	Area	Concentration (µg/ml)	Concentration (µg/g)
Gallic acid	30.83	2.43	36.46
Chlorogenic acid	28.42	4.30	64.52
Catechin	1.49	0.35	5.28
Coffeic acid	2.66	0.20	2.93
Syringic acid	2.17	0.19	2.78
Pyro catechol	16.38	2.38	35.69
Vanillin	41.11	1.42	21.32
Cinnamic acid	42.52	0.85	12.71
Kaempferol	18.02	2.82	42.36

MOS (1g/15 ml)
	Area	Concentration (µg/g)
Vitamin B1	11.65	437.14
Vitamin B2	5.37	76.50
Vitamin B6	35.15	2693.42
Vitamin B9	3.40	244.52
Vitamin B12	12.60	1577.69

Compound	Area (Sample)	Concentration S (ng/g)	Concentration S (mg/g)
ASP	1426.98	10711189.79	10.71
GLU	6742.52	60270978.95	60.27
Serine	1632.96	8117751.79	8.12
Histidine	441.34	5915776.01	5.92
Glycine	3518.41	12872315.78	12.87
Threonine	1051.08	5883521.42	5.88
Arginine	5301.38	38192078.24	38.19
Alanine	2513.66	10227841.74	10.23
Tyrosine	461.20	3846887.14	3.85
Cystine	4.24064	597574.11	0.60
Valine	1817.97	9686963.72	9.69
Methionine	1072.21	5130091.14	5.13
Phenylalanine	1399.57	11036623.03	11.04
Isoleucine	1719.54	8218485.13	8.22
Leucine	2687.61	14500137.30	14.50
Lysine	221.63	3599818.29	3.60
Proline	454.56	10849480.01	10.85

Parameters	Groups
	G1: Adult control (N = 5)	G2: Adult+MOS (N = 5)	G3: Old control (N = 5)	G4: Old+MOS (N = 5)
AST (U/L)	75.6 ± 1.02	72.8 ± 1.01	87.8 ± 2.8	78.2± 8 . 3
Tukey test	a	a	a	a
ALT (U/L)	25.2 ± 2.05	20.0 ± 0.54	40.4 ± 5.19	34.0±2.5
Tukey test	ab	a	c	bc
Albumin (g/dL)	3.0 ± 0.25	3.86 ± 0.13	2.54 ± 0.17	3.68±0. 20
Tukey test	ab	c	a	bc
Total protein (g/dL)	5.98 ± 0.18	6.50±0.266	4.72±0.33	6.90±0.25
Tukey test			a
Urea (mg/dL)	54.8 ± 2.13	46.5±1.6	60.2±1.8	48.96±1.92
Tukey test	bc	a	c	ab
Creatinine (mg/dL)	0.46 ± 0.02	0.34±0.02	0.74±0.05	0.50±0.044
Tukey test	ab	a	c
Free T3 (pg/mL)	5.62 ± 0.36	8.07±0.7	3.66±0.1	6.38±0.47
Tukey test		c	a	bc
Free T4 (ng/dl)	2.10 ± 0.02	3.6±0.31	2.0±0.03	3.1±0.2
Tukey test	a		ab
Free testosterone (pg/ml)	14.3 ± 0.6	19.6±1.2	13.2±0.6	18.7±0.7
Tukey test		a	a	ab

	Testicular			Brain
Oxidative marker Groups	GST (nmol/min/ mg protein)	AChE (µmol/min/ µg protein)	MDA (nmol/mg protein)	GST (nmol/ min/mg protein)	AChE (µmol/min/µg protein)	MDA (nmol/mg protein)
G1: Adult control	176.4 ± 2.83	4.98 ± 0.31	158.6 ± 2.2	235 ± 8.94	5.92 ± 0.24	149.4 ± 1.63
Tukey test
G2: Adult+MOS	251.2 ± 5.49	9.24 ± 0.12	147 ± 1.87	336 ± 9.28	10.82 ± 0.32	134 ± 1.78
Tukey test	d	d	a	c	d	a
G3: Old control	97.0 ± 4.63	2.96 ± 0.166	209.4 ± 2.8	100 ± 20.7	4.12 ± 0.21	187.4 ± 1.63
Tukey test	a	a	d	a	a	d
G4: Old+MOS	215.8 ± 3.89	6.84 ± 0.172	183.8 ± 2.3	288.2 ± 4.8	7.88 ± 0.32	170.2 ± 2.57
Tukey test	c	c	c	c	c	c

Organ	Histopathological alterations	G1: Adult control	G2: Adult+MOS	G3: Old control	G4: Old+MOS
Testes	Degenerated seminiferous tubules	+	-	+++	-
Brain	nuclear pyknosis and neuronal degeneration	+	-	+	-

Results

Discussion

The worldwide issue of population aging is becoming increasingly problematic as the global economy and medical care expand. Age-related disorders are affecting an increasing number of senior individuals25. Consequently, the demand for natural plant-based treatments that prevent and treat age-related illnesses is growing. This study employs MOS to assess its effects as an antioxidant and anti-aging agent against oxidative stress caused by aging, such as brain and testicular damage.

The phytochemical composition, vitamin B content, and amino acids of MOS used in this study are presented in Table 1, Table 2 and Table 3. MOS has been shown to contain bioactive secondary metabolites such as flavonoids (catechin, kaempferol, vanillin), with the most abundant being kaempferol (42.36 µg/g), and phenolic acids (ellagic acid, gallic acid, ferulic acid, chlorogenic acid), with the most abundant being chlorogenic acid (64.52 µg/g). Manisha .26 demonstrated that MO leaves contained flavonoids, mainly quercetin and kaempferol, with concentrations of up to 137.81 and 106.75 mg/g, respectively, and phenolic acids, which include ferulic acid, ellagic acid, and chlorogenic acid.

Infertility affects 50-80 million people worldwide, with males accounting for 20-50% of cases27. In the present study, the effect of MOS in enhancing male reproduction is clearly observed in the aged group compared to the control. The antioxidants in MOS worked in tandem with the epididymis's antioxidant system to further protect and improve the process of spermatogenesis. Aging may be associated with a rise in endogenous ROS, which reduces antioxidant enzyme activity, with Leydig cells being particularly vulnerable to this impact. High ROS levels can alter the hormonal balance that governs male reproductive processes by acting on the hypothalamic-pituitary-gonadotropic (HPG) axis28. Reduced LH secretion causes Leydig cells to generate insufficient testosterone29, resulting in uncontrolled spermatogenesis and suppression of sexual behavior30. In spermatozoa, MDA molecules enter the cell membrane structure, disrupting the symmetric distribution of lipid membrane components. Lipid peroxidation destroys the central region of the sperm cell, resulting in a loss of acrosome ability for fertilization31. Moringa leaves might have beneficial effects on improving male sexual performance in stress-induced sexual dysfunction in rats32. Zade .33 investigated the effects of the aqueous seed extract on the frequency of mounting and intromission in male rats. The investigators concluded that MOS may improve male sexual behavior because it also significantly boosts desire and sperm count. MO has been used to treat male sexual functions such as desire, erectile dysfunction, and testicular damage34. After oral administration of MO, aged rats (18-19 months old) showed improvements in sperm count and morphology, indicating its potential utility in the treatment of sperm abnormalities35. Furthermore, MO is a preventative approach for a variety of ailments and disorders, such as testosterone-induced benign prostatic hyperplasia36.

This study provides findings on the role of MOS on brain cholinergic enzyme (AChE) and MDA. The G2 and G4 groups (MOS administration) presented a significant drop in MDA level and an increase in GST and AChE activities when compared to control groups. MOS contains high phenol and flavonoid content, which are crucial oxidative components in plants. Their antioxidant activity is attributed to their redox characteristics, which help scavenge and neutralize free radicals. MO is utilized to treat neurodegenerative illnesses, including Alzheimer's, ischemic stroke, and epilepsy37. Aging of the brain can cause memory and cognitive impairment, which is commonly associated with changes in the structural flexibility of dendritic spines. In elderly animals and humans, spine number and maturity are reduced, along with alterations in synaptic transmission, which may represent abnormal neuronal plasticity intimately related to reduced brain function. In the most severe cases, a neurodegenerative illness that entirely destroys the basic processes of brain development is possible38.

This study concluded that MOS improves the role of the thyroid by raising thyroid hormones (FT3 and FT4) and reducing oxidative stress in aged male rats when compared to adults. Tahiliani and Kar39 revealed that MO leaf extract has therapeutic efficacy in managing hyperthyroidism, an autoimmune condition, by suppressing T3 production and release.

Our results showed that MOS treatment led to improvements in AST and ALT levels, suggesting a hepatoprotective effect. A modest dose of M. seeds (500 mg/kg B.W.) was more effective at lowering ALT, although larger doses (1000 & 2000 mg/kg B.W.) produced the lowest level when compared to the control group20.

This study concluded that MOS treatment led to improvements in urea and creatinine levels, suggesting a renal protective effect. MO reduces inflammation and oxidative stress, which are common causes of kidney disease40.

Conclusions

From the current study, it is clear that MOS can increase sexual behavior in male rats and could potentially have an impact depending on particular circumstances. MOS exhibited a significant decrease in the MDA level and an increase in GST and AChE activities in brain tissue. Additionally, MOS improves the role of the thyroid by raising the thyroid hormones FT3 and FT4. These findings highlight the crucial role of wild MOS bioactive components in brain, thyroid, and testicular function. Further study in this area may uncover the underlying mechanisms, paving the way for the development of new treatments for age-related issues.

Abbreviations

ABTS: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), AChE: Acetylcholinesterase, ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, DPPH: 2,2-diphenyl-1-picryl-hydrazyl-hydrate, EAEA: Egyptian Atomic Energy Authority, FT3: Free triiodothyronine, FT4: Free thyroxine, GST: Glutathione S-transferase, HPG Axis: Hypothalamic-pituitary-gonadotropic axis, HPLC: High-Performance Liquid Chromatography, LH: Luteinizing hormone, LPO: Lipid peroxidation, MDA: Malondialdehyde, MO: Moringa oleifera, MOS: Moringa oleifera seeds, ROS: Reactive oxygen species

Acknowledgments

The authors wish to extend their heartfelt appreciation to Prof. Dr. Adel Bakeer, an esteemed Professor of Pathology at the Faculty of Veterinary Medicine, University of Cairo, for his invaluable support throughout the histopathological examination.

Author’s contributions

Noha Sayed Hamed: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Formal analysis, Data curation, conducted the experiments, Conceptualization. Hoda Badr Hammad: Editing, Validation, Data curation. Mona Ibrahim Abdou: Writing – review & editing, Formal analysis, Data curation, Conceptualization. All authors read and approved the final manuscript.

Funding

None.

Availability of data and materials

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

Ethical approval was sought and obtained from the Ethical Committee at the National Center for Radiation Research and Technology, EAEA (NCRRT- EAEA-12A/20).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.