Busulfan is a bifunctional alkylating agent widely used in clinical medicine, at low doses in prolonged regimens for chronic myeloid leukemia and ovarian cancer, and at high doses as a conditioning drug before bone marrow transplantation. Its therapeutic power comes with a notorious cost: the drug preferentially destroys rapidly dividing cells, and spermatogonia, the stem cells of sperm production, sit squarely in the blast zone. Previous work has shown that busulfan triggers oxidative apoptosis in spermatogonial stem cells, depletes the germinal epithelium of seminiferous tubules, and can leave cancer survivors with drastically reduced sperm counts. As cancer diagnoses rise globally and survival rates improve, preserving fertility in young male patients has become an increasingly urgent research priority.

Telfairia occidentalis, known locally as ugu, ewuroko, or ikong-ubong depending on the Nigerian region, is an edible leafy cucurbit whose seeds are consumed as snacks, soup condiments, and fermented seasonings. The plant is rich in essential oils, vitamins, minerals, amino acids, flavonoids such as quercetin and kaempferol, alkaloids, carotenes, and cucurbitacines, which together underpin its documented antioxidant, anti-inflammatory, antidiabetic, and antimicrobial properties. Earlier experiments had already hinted at reproductive benefits: fluted pumpkin seed oil reversed alcohol-induced germ cell loss in Sprague-Dawley rats, co-administration of the seeds with caffeine protected the spermatogenesis score index in Wistar rats, and seed fractions attenuated doxorubicin-induced testicular toxicity. The Port Harcourt team set out to dissect which chemical fractions of the seed drive which effects, using two parallel experimental designs over a 54-day treatment period.

The researchers harvested mature fluted pumpkin pods from a local market in Ogoni, Rivers State, verified the plant identity botanically, and macerated the dried, ground seeds in 60 percent aqueous ethanol. After concentrating the crude extract, they split it with a separation funnel into a hexane fraction enriched in non-polar lipids and a freeze-dried aqueous fraction rich in water-soluble compounds. In the injury study, adult male Wistar rats received busulfan at 15 milligrams per kilogram body weight, injected intraperitoneally once weekly for two weeks, alongside oral doses of the hexane fraction at 50, 100, or 200 milligrams per kilogram given twice weekly for the full 54 days. In the companion study, healthy rats received the aqueous fraction three days weekly at the same dose range, with corn oil serving as the vehicle control in both experiments. The 54-day window was chosen to span a full cycle of spermatogenesis, ensuring that any effect on sperm production could manifest.

The biochemical readouts painted a vivid picture of busulfan’s assault and the hexane fraction’s counteroffensive. Busulfan alone significantly shrank testis weight and the gonado-somatic index, spiked lipid peroxidation as measured by malondialdehyde levels, and raised the activities of catalase, superoxide dismutase, glutathione reductase, and glutathione S-transferase, a constellation of changes signaling severe oxidative stress and a disrupted glutathione redox balance. Testicular marker enzymes including acid and alkaline phosphatase and gamma-glutamyl transpeptidase, all of which climb when the seminiferous epithelium degenerates, were also elevated, while lactate dehydrogenase, essential for germ cell energy metabolism, fell. Co-treatment with the hexane fraction reversed virtually every one of these abnormalities in a dose-dependent manner, restoring glutathione status, quenching lipid peroxidation, normalizing enzyme activities, and recovering testis weight toward control levels, with the 200 milligram per kilogram dose showing the greatest efficacy.

Under the microscope, the contrast was equally striking. Control rats displayed intact seminiferous tubules brimming with the full cast of spermatogenic cells and tuffs of mature spermatozoa in the lumen. Busulfan left the tubules vacuolated, distorted, and stripped of spermatozoa and spermatids, a classic signature of maturation arrest. Animals given the hexane fraction showed markedly better tubular architecture, with intact basement membranes lined by spermatogonia and substantially less degeneration, although the authors note that some tubules still contained reduced germ cell layers, indicating partial rather than complete rescue. Stereological analysis with ImageJ software confirmed that the extract prevented the busulfan-induced collapse of tubular diameter and seminiferous epithelial height at all doses tested.

The aqueous fraction told a different, quieter story in healthy rats. After 54 days of treatment, seminiferous tubular diameter, luminal diameter, tubular cross-sectional area, tubular length per gram of tissue, epithelial thickness, and, most intriguingly, the number of testosterone-producing Leydig cells all increased in proportion to dose. Yet the Johnsen spermatogenesis score index and the counts of spermatogonia, spermatocytes, round spermatids, and Sertoli cells remained unchanged, as did body weight, absolute testis weight, and the gonado-somatic index. The researchers interpret this as an enhancement of the testis’s structural and steroidogenic infrastructure, potentially improving sperm passage and androgen synthesis, without an outright acceleration of sperm cell production.

Gas chromatography-mass spectrometry helped explain the divergent behaviors of the two fractions. The hexane fraction yielded 270 identified metabolites, 17 of them abundant, dominated by long-chain fatty acids and related lipids including squalene, n-hexadecanoic acid, octadecanoic acid, conjugated linoleic acid, alpha-linolenic acid, and an ascorbic acid derivative. Many of these lipids are known antioxidants, consistent with the fraction’s ability to blunt busulfan-driven oxidative injury. The aqueous fraction contained 277 metabolites, 14 of them abundant, including glycerin, a compound previously shown to modulate testicular androgen production and testicular morphology. The authors propose that the lipid repertoire underwrites protection against injury, while water-soluble constituents such as glycerin sculpt testicular stereology.

The team is candid about the limits of the work: the two fractions were tested in different animal models rather than head-to-head, and future studies should isolate individual bioactive compounds, test them in additional models of gonadal injury, and evaluate liver and kidney safety markers before any clinical translation. Still, the convergence of biochemical, histological, stereological, and metabolomic evidence makes a compelling case that a common Nigerian food seed contains separable chemical programs, one that shields the testis from chemotherapy’s collateral damage and another that may bolster its hormonal machinery. For a world in which more than a million new cancer cases are diagnosed annually and male infertility is a feared consequence of cure, that duality is precisely the kind of leads translational reproductive medicine has been searching for.

The decision to study seed fractions separately reflects a broader principle in pharmacognosy research. Crude plant extracts contain hundreds of compounds with varying solubilities, and pooling them can obscure which molecules are actually responsible for a given biological effect. By partitioning the aqueous ethanol extract between hexane and water, the Port Harcourt team effectively separated the seed’s lipid-soluble cargo from its water-soluble constituents, allowing each chemical repertoire to be evaluated on its own terms. This approach mirrors earlier work on other edible plants, where non-polar fractions rich in tocopherols, phytosterols, and unsaturated fatty acids often carry antioxidant activity, while polar fractions contribute different bioactivities.

The choice of busulfan as the injury model deserves note. Because the drug reliably depletes spermatogonial stem cells while sparing the somatic framework of the testis, it has become a standard tool for generating reproducible testicular damage in rodents. It is also used experimentally to condition recipients for spermatogonial stem cell transplantation, a technique being explored as a fertility-preservation strategy for prepubertal boys facing gonadotoxic therapy. Any compound that protects the germinal epithelium in a busulfan model is therefore of interest not only as a potential co-adjuvant during chemotherapy but also as a candidate for improving the efficiency of stem cell-based fertility restoration.

The stereological findings in healthy rats also carry implications for how such plant products should be interpreted. An increase in Leydig cell number without a corresponding rise in spermatogenic cell counts suggests that the aqueous fraction may be acting on the interstitial compartment, the site of testosterone synthesis, rather than on the seminiferous tubules directly. Since adequate intratesticular testosterone is required to maintain the blood-testis barrier and support later stages of sperm development, expanding the Leydig cell population could, in principle, strengthen the hormonal support system on which spermatogenesis depends, even if the immediate effect on germ cell numbers is neutral.

It remains important to temper enthusiasm appropriately. Rodent doses of 50 to 200 milligrams per kilogram are far higher, on a body-weight basis, than what a person would obtain from eating fluted pumpkin seeds as food, and the extract was administered as a concentrated fraction rather than a whole seed matrix. Absorption, metabolism, and distribution of the identified lipids and water-soluble compounds in humans may differ substantially from rats. Moreover, the absence of reported toxicity data in this study means that long-term safety, particularly at high doses, has not been established. These caveats notwithstanding, the study illustrates how a detailed fraction-by-fraction dissection of a traditional food plant can yield mechanistically grounded leads for protecting male reproductive health.

Subject of Research: Protective and enhancing effects of Telfairia occidentalis seed extract fractions on rat testicular function and busulfan-induced testicular injury

Article Title: Hexane fraction of Telfairia occidentalis (Cucurbitaceae) ethanol seed extract ameliorates busulfan-induced testicular damage while aqueous fraction enhances testis function in normal rats

Article References: Abarikwu, S. O., Erekeere, C. K., Timi-Johnson, E., Ogbonnaya, A. O., & Ezim, O. E. (2026). Hexane fraction of Telfairia occidentalis (Cucurbitaceae) ethanol seed extract ameliorates busulfan-induced testicular damage while aqueous fraction enhances testis function in normal rats. Discover Toxicology, 3(1), Article 16. https://doi.org/10.1007/s44339-026-00061-1

Image Credits: AI Generated

DOI: 10.1007/s44339-026-00061-1

Keywords: Telfairia occidentalis, fluted pumpkin seeds, busulfan, testicular damage, spermatogenesis, oxidative stress, Leydig cells, seminiferous tubules, male fertility, phytochemicals, testicular stereology, Discover Toxicology