Gas Chromatography-Mass spectrometric (GC-MS) Revealed Bioactive Constituents of Brassica Oleracea var. Viridis (Collard Greens) Used as Ethnomedicine to Treat Male Infertility in Uganda

Introduction

For centuries, medicinal plants have played a vital role in traditional healing systems across cultures, primarily due to their rich content of bioactive compounds.¹ These phytochemicals such as flavonoids, alkaloids, tannins, terpenoids, and saponins have demonstrated significant therapeutic potential.² Acting individually or synergistically, they exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-diabetic, anti-cancer, and reproductive health-enhancing effects.³ Their contribution to drug discovery continues to be profound, as the identification of novel therapeutic agents often begins with the screening of natural products from diverse plant sources.⁴

Brassica oleracea var. viridis (commonly known as collard greens or “Sukuma wiki” in Swahili) is a nutrient-rich leafy vegetable widely cultivated and consumed in Uganda and other parts of East Africa as shown in Figure 1. Characterized by large, broad leaves ranging in color from deep green to bluish-green, collard greens thrive in a variety of climates and are valued both as a food source and in ethnomedicine. Traditionally, this plant has been used to manage various health conditions, including male reproductive issues, though its phytochemical basis remains underexplored.

Figure 1. Diagram of Brassica oleracea var. viridis (collard green)¹⁸.

The health-promoting properties of collard greens are largely attributed to their high content of vitamins C, E, and K, dietary fiber, calcium, and a diverse range of bioactive compounds. These constituents support immune function, bone health, digestion, and oxidative balance.⁵ Additionally, preliminary evidence suggests potential anti-hyperglycemic and fertility-enhancing effects, reinforcing its traditional use in managing male infertility.

Gas Chromatography-Mass Spectrometry (GC-MS) is a robust and precise analytical technique employed in phytochemical investigations to identify and quantify chemical constituents in complex plant matrices.^6,7 This method is especially effective in uncovering bioactive compounds that may explain the therapeutic value of traditionally used plants.

This study aims to analyze the bioactive constituents of Brassica oleracea var. viridis collected in Uganda using GC-MS. By identifying these compounds, the research seeks to provide scientific validation for the ethnomedicinal use of collard greens in the treatment of male infertility and contribute to the growing body of knowledge on plant-based fertility therapies.

Methodology

Results

Phytochemical screening of the Ethanol extraction of Brassica oleracea var. viridis (Collard Greens) leaves revealed the presence of the phytochemical constituent’s flavonoids, alkaloids, phenolic compounds, fatty acids, terpenoids, and other compounds as detailed in Table 2.

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.

Discussion

Phytochemical screening of the ethanol extract from Brassica oleracea var. viridis (Collard Greens) has revealed a diverse array of phytoconstituents with potential health benefits, particularly in relation to reproductive health, as summarized in Table 4. Among the identified compounds, Phytol, a terpenoid, stands out due to its notable antioxidant properties. Previous studies have demonstrated that Phytol exhibits significant antioxidant activity in vitro, including scavenging hydroxyl radicals and nitric oxide, as well as inhibiting the formation of thiobarbituric acid reactive substances (TBARS).¹¹ These findings are consistent with a systematic review by,¹² which linked antioxidants to the restoration of oxidative stress-induced sperm abnormalities. However, it is important to acknowledge the contrasting results presented by,¹³ who showed that antioxidant supplementation might not enhance semen parameters or DNA integrity in men with male factor infertility. This highlights the complexity of antioxidant supplementation in fertility therapies and suggests that a more nuanced approach may be necessary.

Additionally, the screening identified several omega-3 fatty acids, including 9,12,15-octadecatrienoic acid (Z,Z,Z), pentadecanoic acid, and 7,10,13-hexadecatrienoic acid (Z,Z,Z). These compounds are well-documented for their beneficial effects on sperm quality, motility, and overall reproductive health. A study by¹⁴ indicated that omega-3 supplementation could enhance antioxidant activity in human seminal fluid, leading to improvements in sperm count, motility, and morphology. This is further supported by a systematic review and meta-analysis conducted by,¹⁵ which found that omega-3 supplementation significantly improved sperm motility, increased docosahexaenoic acid (DHA) levels in seminal plasma, and enhanced total sperm count and cell density.

The presence of phenolic compounds and flavonoids in the B. oleracea var. viridis extract also warrants attention, given their potent antioxidant properties. In particular, 4-ethenyl-2,6-dimethoxyphenol has been noted for its potential neuroprotective effects, which may have implications for psychogenic male infertility.¹⁶ Furthermore, Gamma-Sitosterol, a plant sterol, exhibits both antioxidant and anti-inflammatory activities, potentially promoting male fertility through modulation of oxidative stress and inflammatory pathways. Research by¹⁷ on β-sitosterol further supports its role in inhibiting the growth and spread of prostate cancer cells, which could indirectly enhance reproductive health by preserving prostate function.

In conclusion, the diverse phytochemicals present in Brassica oleracea var. viridis (Collard Greens) including Phytol, omega-3 fatty acids, phenolic compounds, flavonoids, and sterols suggest significant potential benefits for male reproductive health. These compounds appear to exert their effects through antioxidant, anti-inflammatory, and potentially neuroprotective pathways. 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.

Conclusion

In conclusion, the phytochemicals identified in Brassica oleracea var. viridis (Collard Greens), including Phytol, Omega-3 fatty acids, phenols, flavonoids, and sterols, demonstrate potential benefits for male reproductive health, particularly through their antioxidant, anti-inflammatory, and possibly neuroprotective effects. While studies show positive associations between these compounds and improvements in sperm quality, motility, and overall reproductive health, the contradictory findings regarding antioxidants, such as their limited impact on semen parameters in some studies, highlight the complexity of their role in fertility therapy. Consequently, further research is needed to clarify the effectiveness of these bioactive compounds in clinical settings and to establish concrete guidelines for their use in treating male infertility.

Declarations