The free radical scavenging capacity of MOS was determined using the stable DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), according to Hwang and Do Thi19.

ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) stock solutions were produced according to Hwang and Do Thi19.

The phytochemical, amino acids, and vitamin B composition of MOS were analyzed using HPLC. Fifty milligrams of MOS was combined with 5 mL of H₂O and 5 mL of HCl, heated at 100°C for 24 hours, and filtered. Finally, 1 mL of filtrate was dried, resuspended in 0.1 M HCl, and injected into the HPLC. HPLC analysis was performed using an Agilent 1260 series. The Eclipse C18 column (4.6 mm x 250 mm i.d., 5 μm) temperature was maintained at 40°C.

Dry Moringa oleifera seeds (MOS) were purchased from the laboratories of the National Research Centre, Giza, Egypt. MOS were washed and peeled. A mortar was used to grind and finely powder the seeds, which were then used as a suspension in distilled water. Twenty male Wistar albino rats were obtained from the animal house of the Nuclear Research Centre, Egyptian Atomic Energy Authority (EAEA). Before beginning the experiment, rats were kept in the Animal House of the Nuclear Research Centre's Radioisotopes Department for two weeks.

Rats were divided into four groups (five animals each): G1: five normal adult rats (2 months) without any treatment. G2: five normal adult rats (2 months) received orally 500 mg/Kg b.wt MOS (in a gradual increase of the dose from 100-500 mg/Kg b.wt during the first week) for 4 weeks (five days/week) according to El-Hak et al.20, with some modification. G3: five aged (14 months) male rats without any treatment. G4: five aged (14 months) male rats received orally 500 mg/Kg b.wt MOS as in G2.

Twenty-four hours after the last administration of MOS, we anesthetized the rats and humanely decapitated them to collect samples. Blood samples were collected without anticoagulants and centrifuged for five minutes at 3000 rpm to separate the serum. The rats' brain and testicular tissues from each group were subsequently collected.

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) enzymes, albumin, urea, and serum creatinine were determined using assay kits obtained from Vitro Scient Co. (Cairo, Egypt).

Serum FT3 (Cat. No. A13430), FT4 (Cat. No. 33880), and free testosterone (Cat. No. DSL4900) were determined using a commercial kit purchased from Beckman Coulter, Immunotech, Czech Republic.

The brain and testicular tissues of the rats in each group (10% wt/v) were promptly homogenized using a German IKA homogenizer. The tissue was cleansed with ice-cold PBS (0.01M, pH 7.4). Then, the homogenates were centrifuged at 5000 rpm for 15 minutes at 4°C with a Beckman Coulter Allegra 64R fixed-angle rotor (PN 392050).

Acetylcholine Esterase (AChE): AChE activity was measured according to Ellman et al.21.

Glutathione S-transferase (GST) Kinetic Assays: GST activities towards 1-chloro-2,4-dinitrobenzene (CDNB) were measured spectrophotometrically at 340 nm according to Habig et al.22.

Lipid Peroxidation: Lipid peroxidation (LPO) was assessed in terms of malondialdehyde (MDA) creation, according to the assay stated by Ohkawa et al.23. The MDA concentration was quantified as nmol MDA/mg protein.

The brain and testicular tissue samples were collected in various groups and well-preserved in 10% formalin saline for one day. After washing with tap water, dehydration was accomplished using successive dilutions of alcohol (methyl, ethyl, and absolute ethyl). Specimens were cleaned in xylene and fixed in paraffin at 56°C in a hot air oven for one day according to Banchroft et al.24.

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.

Table 1

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

Figure 1

Table 2

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

Figure 2

Table 3

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

Table 4

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

Data expressed as mean ± SE, n = 5. Values with the same superscript in the raw are not statistically different. The groups are statistically significant (P < 0.05) as compared with control; using one-way ANOVA followed by Tukey’s HSD multiple comparisons as a post-hoc test.

Table 5

	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

Data expressed as mean± SE, n = 5. Values with the same superscript in the column are not statistically different. The groups are statistically significant (P < 0.05) as compared with control; using one-way ANOVA

Table 6

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	+	-	+	-

+++ sever, ++ moderate, + mild, – nill

Figure 4

Figure 5

The in vitro results indicated that DPPH scavenging activity had a value of 0.395 mg trolox/g, and the radical scavenging activities of ABTS were 0.802 mg trolox/g.

The phytochemical, amino acids, and vitamin B of MOS were estimated using HPLC. The individual phenolic and flavonoid compounds results showed that chlorogenic acid and kaempferol had a strong response and more intense peaks (Table 1 and Figure 1). The vitamin B content in MOS was estimated using HPLC. The results showed that vitamins B6 (2693.42 µg/g) and B12 (1577.69 µg/g) had the highest concentration, as shown in Table 2 and Figure 2. The amino acid content in MOS is shown in Table 3 and Figure 3.

Acute oral administration of MOS up to 500 mg/kg resulted in no adverse effects in the experimental animals, including food intake, atypical body growth, decreased activity, diarrhea, bleeding, or mortality. As a result, no fatal dosage was established in this study.