Investigating the Impact of Launaea angustifolia Extract on the Reproduction Function of Female Rats

Recently, there has been a growing trend among people from various nations to utilize traditional medicine as a supplementary or alternative approach for addressing common health conditions (Yekta et al., 2023; Jamal, 2023). There are numerous medicinal plants that are used as dietary supplements or biopharmaceuticals instead of manufactured drugs. Nevertheless, a considerable number of these medicinal plants necessitate additional scrutiny regarding their adverse effects and examination of their constituents (Goshtasbi et al., 2023). The characteristics of phytochemical constituents range significantly among various species. The Launaea genus, belonging to the Asteraceae family, encompasses a total of 55 to 59 species distributed over several countries in the southern and western regions of the Mediterranean, as well as in North Africa and Southwest Asia.

Launaea comprises a total of forty species that possess medicinal capabilities, including the ability to alleviate stomachache, treat skin illnesses, combat cardiovascular diseases, lower cholesterol levels, fight against cancer, inhibit microbial and fungal growth, reduce inflammation, function as antioxidants, exhibit cytotoxic effects and possess neuropharmacological properties (Marzouk et al., 2021; Michel et al., 2020). Moreover, the anti-diabetic properties of the ethanolic extract of Launaea nudicaulis in rodents induced with streptozotocin have been attributed to the flavonoid and phenolic compounds it contains (Kumer et al., 2023). Moreover, the anti-diabetic properties of the ethanolic extract of Launaea nudicaulis in rodents induced with streptozotocin have been attributed to the flavonoid and phenolic compounds it contains (Khan et al., 2023), while reducing by the activation of oxidative defense mechanisms, alloxan-induced pathological consequences (Gbadamosi et al., 2020). Moreover, El-Fadaly et al.  (2023), the researchers documented the capacity of Launaea mucronata extract to enhance biochemical indicators of renal function in a chromium-treated rodent model (K2Cr2O7). In particular, both male and female rodents exhibited a protective effect against hepatic enzymes and kidney functions when exposed to Launaea angustifolia (Aiman et al., 2023). Despite therapeutic and toxicological studies using Launaea sp. extracts, their effects on reproductive physiology in male and female rats have not been studied until now. Therefore, the primary objective of this study is to investigate the hormonal and histological transformations in the reproductive organs of rats after the administration of Launaea angustifolia methanol extract.

Chemicals
 
Unless otherwise specified, all chemicals utilized in this undertaking were acquired from Sigma.
 
Plant collection and extraction
 
The Launaea angustifolia plant was collected during the time from 1^st Feb to 1^st April 2022, from the Al-Thumamah area (25.25141°N 46.63067°E), Riyadh city. The leaves were removed from the plant, washed and kept in a dark and cleaned place until dried. Then, the leaves were grained until they became powdered. The crushed plant leaves of Launaea angustifolia were soaked in methanol for a day. Later, the solution was filtered via the Whatman paper. After maceration, the Launaea angustifolia extract was evaporated at a temperature between 50 and 60°C, then the powder was frozen at -20°C until the dosing preparation. Before the trial, one hundred milligrams of the Launaea angustifolia extract was dissolved in DMSO to prepare the concentrations (10, 50 and 100 mg/kg of rat) required for dosing (Alhimaidi et al., 2022).
 
Experimental design
 
female rats in good health were obtained from the animal facility at the Zoology Department of the Science College, King Saud University (KSU). The rats were accustomed to a well-ventilated area with a suitable room temperature of 25±2°C, a regular light and dark schedule of 12 hours each and provided with a standard diet and water. All experimental procedures were conducted in compliance with the guidelines specified by the ethics committee (Approval no: KSU-SE-23-06) and the Institutional Animal Care at KSU. The project was conducted on female rats (25 rats / female), with an average weight 200-240 g and ages between 12 and 15 weeks. Each female of rats was divided separately into five groups (5 rats / group). The G-control was without any dosage, while the G-negative control was treated by oral gavage with DMSO. The G-10, G-50 and G-100 were given 10, 50 and 100 mg/kg bw/day respectively of Launaea angustifolia extract for 14 days. 
 
Biochemical analysis
 
At day 15, Blood was drawn from animal heart in non-heparinized tubes, cooled at ~4°C for overnight, then centrifuged at 1000x g for 15 min to get serum and frozen at -20°C up to hormonal studies. The enzyme-linked immunoassay (ELISA) method was used to estimate all hormone concentrations in serum samples by the following ELISA kits (MOLEQULE-ON company, Auckland, New Zealand): 17 β-estradiol (Cat No. ELI-M-036-96), Progesterone (Cat No. ELI-M-034-96).
 
Histomorphometric examination
 
The ovary were collected after the animals were anesthetized with CO2 and socked in 10% neutral formalin for 48 hours for fixation. The fixed gonads were dehydrated by gradual alcoholic solutions, infiltrated in xylene for 4-6 hr., impeded in paraffin wax cubes, sectioned by microtome, mounted on slides and stained with H&E. The histomorphological and histomorphometric evaluations were examined by a light microscope. Ovarian follicles were counted and classified in all animal groups according to Adelakun et al., (2022) with some modifications. Primordial Follicles were identified by the patchy row of squamous cells around the oocyte. Primary and secondary follicles were recognized by the presence of one row and two rows of cuboidal cells respectively. The tertiary follicles were distinguished by the presence of numerous layers and small antrum. Graafian follicles were characterized by a large follicular antrum.
 
Statistical analysis
 
All statistics obtained from this experiment were analyzed by the GraphPad Prism (10.1.0) program. The Shapiro-Wilk test demonstrated the normal distribution of our data. The animals body weights were analyzed using two-way ANOVA (day factor × extract factor) and Tukey’s multiple comparisons test. Hormone levels, weights of the reproductive organs data were examined using one-way ANOVA, followed by Tukey’s test. The outcomes are presented as the average values and standard deviations and any P value less than or equal to 0.05 indicates a significant finding.

Although the body weights of female rats increased during this study, there were no significant differences between groups after 14 days of dosing (Fig 1).



Female rats, the body weights were significantly higher (P<0.05) in all experimental days (except day 1 and day 3) in the control group compared to the 50 and 100 mg groups. While there was a significant increase in ovarian weights in the control group compared to the DMSO group (Fig 2).

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17 β-estradiol and progesterone levels in all female after the end of the experiment are shown in Fig 3, 4. There were no statistically significant differences between the concentrations of 17 β-estradiol and progesterone in the female groups under study.

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Percentages of ovarian follicles in their different stages there are significantly different in treated groups 10 g and 50 g (follicle stage) compared to control group at P<0.05 (Fig 5).

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The scientifically validated approach to identify pharmaceuticals or herbal medicinal formulations involves conducting safety evaluations on different experimental animals prior to their use in humans. The Launaea genus plant is widely recognized for its extensive use in traditional medicine for the treatment of many ailments.

The objective of our inquiry is to examine the impact of L. angustifolia extract chemical screening on the reproductive function of female rats. The findings of our study validated that the L. angustifolia extract, which contained significant levels of fatty acids, significantly improved reproductive functions in female rats that were subjected to the extraction.

Upon doing a chemical examination on the extract of L. angustifolia, it was discovered that the presence of Oleic acids, Linoleic acid, n-Hexadecanoic acid and Octadecanoic acid, along with other phytochemicals, was present in a minor quantity.

A group of researchers who analyzed the chemical makeup of Launaea resedifolia and found that it included 19 compounds, the most of which were dominated by dioctyl phthalate oil, with the majority of the chemical linkages being esters, alcohols, ketones and terpenes. Cheriti et al., (2006) observed that the oil content of L. angustifolia and that of L. arboresens were quite similar to one another. This was also the case when comparing the two species.

There is a possibility that the variation is attributable to the geographical dispersion as well as the time of collection. In contrast, the essential oil of Launaea lanifera, which was investigated by Benmeddour et al., (2015), was distinct from the essential oil of L. angustifolia species. According to Benmeddour et al., (2015) and Pavia et al., (2015), the absence of a distinctive aroma in the L. lanifera oil was related to the fact that the plant material utilized in the L. lanifera oil extraction process comprises a significant quantity of flowers. Additionally, the plant material does not contain any aromatic chemicals.

The findings of this experiment demonstrated that the significant quantity of fatty acids included in the extract of L. angustifolia associated with an improvement on the reproductive function of female rats that were exposed to the extract, particularly at high doses. It is possible that the L. angustifolia extract, which has been shown to have medicinal and therapeutic benefits, might be a strong candidate for the discovery of further pharmacological qualities.

The authors sincerely acknowledge the Researcher Support Project (RSP-2024 R232) for funding this work at King Saud University, Riyadh, Saudi Arabia.

The data sets used in the current study are available from the corresponding author upon request.

All experimental procedures were conducted in compliance with the guidelines specified by the ethics committee (Approval no: KSU-SE-23-06) and the Institutional Animal Care at KSU.

Not applicable.

Not applicable.

The authors declare no conflicts of interest.