Gas Chromatography-Mass spectrometry (GC-MS) Revealed Bioactive Constituents of Brassica oleracea var. viridis (Collard Greens) Used to Treat Male Infertility in Uganda.

Chemicals

Only analytical-grade chemicals and reagents were used for the study.

Plant collection and identification

Fresh leaves of Brassica oleracea var. viridis (collard greens) were collected from Bwejuragye in Ishaka Town, Bushenyi, Igara County, Western Uganda with GPS coordinates of approximately 0°32′42.0″S 30°08′18.0″Ephyt. The plant was identified and authenticated by Dr. Eunice Olet of the Department of Botany, Faculty of Biological Sciences, Mbarara University of Science and Technology. A voucher specimen was deposited in the university’s herbarium under the reference number IOE-24-001. Details of the plant collection and identification are presented in Table 1.

Preparation of plant material

Fresh leaves (2 kg) of Brassica oleracea var. viridis (collard greens) were collected from fields located in Bwejuragye, Bushenyi Local Government Area, Uganda. The samples were subsequently transported to the herbarium unit at Mbarara University for proper botanical identification and authentication. The leaves were thoroughly washed with distilled water to remove surface contaminants such as dirt and sand, and then shed-dried at ambient temperature of 26°C. After complete drying, the leaves were finely ground into a powder and stored in airtight containers in a dry environment for future extraction.

The extraction process was performed using a modified version of the method described by Abdulrahman et al.¹⁰ A total of 800 g of the powdered leaves was cold macerated in 5 liter of 99% ethanol (1:5 w/v) for 72 hours in sterile glass container. The mixture was kept in the dark to prevent degradation at room temperature (26± 2°C) with intermittent shaking every 6-12 hours to enhance solute diffusion and extraction efficiency. The resulting ethanolic mixture was then filtered through sterile Whatman No. 1 filter paper. The filtrate was concentrated to dryness reduced pressure using a rotary evaporator below 40°C having a constant weight. The dried crude extracts were stored at 4°C in an amber glass bottle that was tightly sealed until further analysis. Approximately 5 g of the ethanol extract was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis, and the remaining extract was stored in a refrigerator for subsequent use in further analyses.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis

GC-MS analysis was performed using a Shimadzu gas chromatograph-mass spectrometer.^11,12 Chromatographic separation was achieved with a DB-5 fused-silica capillary column in inert MSD mode, equipped with a Triple-Axis detector. High-purity helium (99.999%) served as the carrier gas at a constant flow rate of 1.0 mL/min. The system was operated under the following conditions: ion source temperature at 250 °C, interface temperature at 300 °C, pressure at 16.2 psi, and out time of 1.8 mm. A 1 μL sample was injected in split mode (1:50) at 300 °C.

The column temperature program began at 36 °C (held for 5 min), ramped to 150 °C at 4 °C/min, then to 250 °C at 20 °C/min, and was held for 5 min, resulting in a total run time of 47.5 minutes. Relative percentage abundances were determined by comparing individual peak areas to the total chromatogram area. Data acquisition and instrument control were managed using MS Solution software, and compound identification was based on mass spectral comparisons with the NIST20 library.

Gas Chromatography-Mass Spectrometry (GC-MS) profiling of ethanol extract of Brassica oleracea var. viridis (Collard Greens)

Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the ethanol extract obtained from the leaves of Brassica oleracea var. viridis (collard greens) identified a total of 77 distinct phytochemical constituents exhibiting a broad spectrum of bioactive properties. The resulting chromatogram is presented in Figure 2, while Table 3 details the identified compounds, including those meeting the inclusion threshold of ≥1.90% area percentage. Compounds with an area percentage below this threshold were excluded from further analysis. The table also provides information on each compound’s retention time (RT), molecular formula, molecular weight (MW), and chemical structure.

Figure 2. Showing the chromatograph of ethanol extract of Brassica oleracea var. viridis.

Table 3. Selected by %age area (1.90 upward for inclusion criteria, exclusion is 1.90 downwards) of ethanol extract of Brassica Oleracea var viridis (collard greens).

S/N	Name	MF	MW	RT	Area%	Structure of compounds
1	1-(cyclopropyl carbonyl) piperidine-3-amine	C9H16N2O	168.24g/mol	9.021	2.33
2	6-Isobutyryl-2,2,4,4-tetra methylcyclohexane-1,3,5-trione	C14H20O4	252.31g/mol	9.280	3.68
3	Phenol, 4-ethenyl-2,6-dimethoxy-	C10H12O3	180.2g/mol	9.508	1.90
4	2,2,4,4-Tetramethyl-6-(2-methyl butanol) cyclohexane-1,3,5-trione	C15H22O4	266.33g/mol	10.080	5.90
5	Bacteriochlorophyll-c-stearyl	C52H72MgN4O4−2	841.5g/mol	10.170	3.71
6	Dihydroisoobtusilactone	C17H28O3	280.408g/mol	10.943	3.23
7	(7Z,10Z,13Z)-hexadeca-7,10,13-trienoic acid	C16H26O2	250.38g/mol	13.387	6.49
8	Pentadecanoic acid	C15H30O2	242.4g/mol	13.623	7.96
9	Phytol	C20H40O	296.5g/mol	14.949	4.72
10	(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid	C18H30O2	278.4g/mol	15.306	13.44
11	Dichloroacetic acid, tridec-2-ynyl ester	C15H24Cl2O2	307.3g/mol	15.452	3.00
12	2,3-dihydroxypropyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate	C21H36O4	352.5g/mol	19.817	2.11
13	Hexatriacontane	C36H74	507g/mol	21.229	3.46
14	gamma.-Sitosterol	C29H50O	414.7g/mol	25.056	3.25

Among the identified compounds are several notable bioactive constituents, including Dimethyl trisulfide, 2-Piperidinemethanamine, 2,7-Dimethyl-1,7-octadien-3-amine, Oxalic acid cyclohexyl propyl ester, 2-Pyrrolidinone, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, and 2,2-Difluoroethylbenzene. Others include 3,4-Anhydro-d-galactosan, dl-trans-Chrysanthemic acid, Phenol derivatives, Indole, 2-Methoxy-4-vinylphenol, and various pyrrolidine and cyclohexane derivatives.

Further constituents of pharmacological interest include 7,10,13-Hexadecatrienoic acid (Z,Z,Z), 9,12,15-Octadecatrienoic acid methyl ester, phytol, γ-tocopherol, α-tocospiro A and B, stigmasterol, ergost-5-en-3-ol (3β), and γ-sitosterol. Many of these compounds have been previously reported to exhibit antioxidant, anti-inflammatory, antimicrobial, and reproductive health-supportive properties, which may explain the traditional use of collard greens in managing male infertility.

The identified bioactive molecules span multiple chemical classes, including fatty acid esters, phenolics, alkaloids, terpenoids, sterols, and tocopherols, underscoring the pharmacological potential of Brassica oleracea var. viridis. Selected compounds and their proposed biological pathways relevant to male reproductive health are illustrated in Figure 3.

Figure 3. Showing the selected potential compounds and their biosynthesis pathways.

Recommendation

Future research should focus on elucidating the specific mechanisms by which these compounds interact and contribute to male fertility, as well as assessing their clinical efficacy and safety for therapeutic use in reproductive health. These trials should specifically assess the effect of these phytochemicals such as phytol, omega-3 fatty acids, phenols, flavonoids, and sterols on sperm quality, motility, and overall fertility outcomes to establish a clearer understanding of their clinical potential of these phytochemicals in treatment of male infertility.

Ethics approval

The authors confirm that all guidelines set by the University’s research ethics for plant collection, characterization, and documentation was duly followed. The plant specimen was identified by the Department of Botany, Faculty of Science, Mbarara University of Science and Technology, Uganda. The experimental protocols received approval from the Kampala International University Research Ethics Committee. The plant collection process adhered to local guidelines and does not require further confirmation.