Graptophyllum pictum (Acanthaceae) relieves some hallmarks of endometriosis in an experimental model in Wistar rats

This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.

Out of the 80 rats introduced in the experiment, three rats died as a result of complications arising from the autotransplantation of the uterus fragment, and two others died following the second operation to assess the success of the transplant. Among the 75 remaining rats, 73 developed endometriotic implants. The others were excluded from the experiment. An induction yield of 91.25% was obtained. The latency time and frequency of writhing in controls and G . pictum extract-treated animals evaluated for 30 min are shown in Fig. 1 . The latency time was higher in the normal control group ( P < 0.05) compared to the SHAM group. After injection of a 2 IU dose of oxytocin, the latency time to the first writhe within 30 min was significantly longer in the SHAM group ( P < 0.05) and Aspirin (ASP at 3 mg/kg body weight (BW); P < 0.01) groups compared to the negative control group (TNEG). Similarly, administration of aqueous and methanolic extracts of G. pictum significantly ( P < 0.001) increased writhing latency in animals in the GPC 257 mg/kg BW, GPM 50 mg/kg BW, and GPM 275 mg/kg BW groups. For writhing frequency, a significant reduction ( P < 0.05) in writhing frequency was observed in the letrozole (LET at 10 mg/kg BW, P < 0.05), aspirin (ASP at 3 mg/kg BW, P < 0.01), and GPM 50 mg/kg BW ( P < 0.01) groups, compared with the TNEG group. Effects of G. pictum on latency (A) and twisting frequency (B). Each value represents the mean ± MSE with n = 5. Control, SHAM, and TNEG animals receiving dilution solvent; LET: animals receiving letrozole at a dose of 10 mg/Kg BW/day; ASP: animals receiving aspirin at a dose of 3 mg/Kg BW/day; GPC: animals treated with aqueous extract of G. pictum at doses of 50 and 275 mg/Kg BW/day; GPM: animals treated with methanol extract of G. pictum at doses of 50 and 275 mg/Kg BW/day. # P < 0.05; ## P < 0.01 corresponds to the degree of significance compared with the SHAM group. * P < 0.05; ** P < 0.01, *** P < 0.001 corresponds to the degree of significance compared with the TNEG group. Table 1 below shows the effects of G . pictum on endometrial implant volume after 7 days of treatment. According to this table, the treatment induced a significant decrease ( P < 0.05; −129.71%) in endometrial implant volume in the GPM 50 mg/kg b.w. group, compared to the TNEG group. However, compared with the negative control group, aspirin, and all G. pictum methanolic and aqueous extracts reduced the percentage variation in endometrial implant volume at all doses. Effects of G . pictum on implant volume after 7 days of treatment. Each value represents the mean ± SEM with n = 5. CONTROL, SHAM, and TNEG animals received dilution solvent; LET: animals received letrozole at a dose of 10 mg/Kg BW/day; ASP: animals received aspirin at a dose of 3 mg/Kg BW/day; GPC: animals treated with aqueous extract of G. pictum at doses of 50 and 275 mg/Kg BW/day; GPM: animals treated with methanol extract of G. pictum at doses of 50 and 275 mg/Kg BW/day. P < 0.05, corresponds to the degree of significance compared with the TNEG group. The effects of G . pictum extracts (aqueous: GPC and methanolic: GPM) on relative ovarian mass after 7 days of treatment are shown in Fig. 2 . The results show that a significant ( P < 0.05) increase in relative ovarian mass was observed in the normal control group compared to the SHAM group. The relative ovarian mass was increased at all the doses with the aqueous and methanolic extracts of G . pictum (GPC 50 mg/Kg BW, P < 0.001; GPC 275 mg/Kg BW, P < 0.001; GPM 50 mg/Kg BW, P < 0.01; and GPM 50 mg/Kg BW, P < 0.01), compared to the negative control group (TNEG). This parameter also increased significantly in the letrozole (LET, P < 0.01) and aspirin (ASP, P < 0.05) groups. Effects of G. pictum on relative uterine and ovary mass, size of uterine epithelium, and microphotographs (×100, hematoxylin and eosin staining) of the experimental rat uterus (C). Each value represents the mean ± MSE with n = 5. Control, SHAM, and TNEG animals receiving dilution solvent; LET: animals receiving letrozole at a dose of 10 mg/Kg BW/day; ASP: animals receiving aspirin at a dose of 3 mg/Kg BW/day; GPC: animals treated with aqueous extract of G. pictum at doses of 50 and 275 mg/Kg BW/day; GPM: animals treated with methanol extract of G. pictum at doses of 50 and 275 mg/Kg BW/day. # P < 0.05; corresponds to the degree of significance compared with the SHAM group. * P < 0.05; ** P < 0.01, *** P < 0.001 corresponds to the degree of significance compared with the TNEG group. Ep: uterine epithelium; Lu: lumen; St: stroma. Regarding the effects of G . pictum extracts (aqueous: GPC and methanolic: GPM) on relative uterine mass and size of eutopic endometrial epithelium, Table 2 shows that aqueous and methanolic extracts of G . pictum did not induce significant variations in the relative mass of the uterus and the size of its epithelium. However, there was a significant increase ( P < 0.05) in these parameters in the normal control group compared to the SHAM group. Effects of G . pictum on some fertility parameters. The results of the oxidative stress markers are shown in Fig. 3A, B, C, D . Serum levels of reduced glutathione (GSH) were significantly ( P < 0.05) decreased in animals in the negative control group (TNEG) compared to those in the SHAM group; however, these parameters were significantly ( P < 0.001) increased with the administration of aspirin and GPC at the dose of 275 mg/Kg BW compared to the TNEG group. Similarly, the serum level of superoxide dismutase (SOD) units was significantly ( P < 0.05) increased in the animals receiving G . pictum extracts (GPC at all doses and GPM 50) compared to the negative control group. In addition, these SOD levels decreased by 19.82%, from 573.65 ± 98.43 Mmol/g of implant in the negative control group to 715.48 ± 50.64 Mmol/g of implant in the SHAM group, although this difference was not statistically significant. Serum nitrite levels were significantly higher ( P < 0.05) in the negative control group (TNEG) than in the SHAM group. On the other hand, administration of the positive controls (letrozole and aspirin) and G . pictum extracts at all doses favored a significant decrease in serum nitrite levels (for ASP; P < 0.05; and P < 0.001 for LET, GPC, and GPM at all doses (50 and 275 mg/kg BW). Effects of G. pictum on oxidative stress markers (GSH, SOD, MDA, NO 2 – ). Each value represents the mean ± MSE with n = 5. Control, SHAM, and TNEG animals receiving dilution solvent; LET: animals receiving letrozole at a dose of 10 mg/Kg BW/day; ASP: animals receiving aspirin at a dose of 3 mg/Kg BW/day; GPC: animals treated with aqueous extract of G. pictum at doses of 50 and 275 mg/Kg BW/day; GPM: animals treated with methanol extract of G. pictum at doses of 50 and 275 mg/Kg BW/day. # P < 0.05; corresponds to the degree of significance compared with the SHAM group. * P < 0.05; ** P < 0.01, *** P < 0.001 corresponds to the degree of significance compared with the TNEG group. Compared with animals in the negative control group, serum malondialdehyde (MDA) levels were significantly reduced in animals treated with methanolic extract of G . pictum at all doses (50 mg/Kg BW, P < 0.01 and 275 mg/Kg BW, P < 0.05) ( Fig. 3 ). However, compared to animals in the SHAM group (2.55 ± 0.24 Mmol/g of implant), a non-significant increase of 44.70% in this parameter was observed in the TNEG group (3.69 ± 0.38 Mmol/g of implant). Figure 4A, B, C shows the concentrations of pro-inflammatory cytokines after 7 days of treatment. Compared to the SHAM group, the negative control group (TNEG) showed maximal concentrations ( P < 0.001) of the pro-inflammatory cytokines, IL-6, TNF-α, and VEGF. However, administration of G . pictum extracts resulted in a significant ( P < 0.001) decrease in the concentrations of all these cytokines in the LET, ASP, GPC 275 mg/Kg, GPM 50 mg/Kg, and 275 mg/Kg groups. Effects of G. pictum on some pro-inflammatory cytokines: IL-6 (A), TNF-, (B), and VEGF (C). Each value represents the mean ± MSE with n = 5. Control, SHAM, and TNEG animals receiving dilution solvent; LET: animals receiving letrozole at a dose of 10 mg/Kg BW/day; ASP: animals receiving aspirin at a dose of 3 mg/Kg BW/day; GPC: animals treated with aqueous extract of G. pictum at doses of 50 and 275 mg/Kg BW/day; GPM: animals treated with methanol extract of G. pictum at doses of 50 and 275 mg/Kg BW/day. ### P < 0.001 corresponds to the degree of significance compared with the SHAM group. * P < 0.05; *** P < 0.001 corresponds to the degree of significance compared with the TNEG group. Figure 5 below shows the effects of G. pictum on folliculogenesis or follicle population in animals treated for 7 days. Compared to the SHAM group, the number of primary follicles increased ( P < 0.05) in the normal control group. Treatment with 50 mg/Kg aqueous extract (GPC) resulted in a significant increase ( P < 0.05) in the number of antral follicles. Treatment with a dose of 50 mg/Kg of methanol extract induced an increase in the number of Degraaf follicles ( P < 0.05) compared to the TNEG group. The number of corpora lutea was increased in the normal control group ( P < 0.05) compared to the SHAM group. However, this parameter was decreased in the negative group ( P < 0.05) compared to SHAM. Compared with the TNEG group, the number of corpora lutea was increased in the LET ( P < 0.01), GPC 50 mg/kg ( P < 0.01), GPM 50 mg/Kg ( P < 0.01), and GPM 275 mg/Kg ( P < 0.05) groups. Effects of G. pictum on the follicular population after a 7-day treatment and microphotographs (X 25, hematoxylin and eosin staining) of rat ovaries. Each value represents the mean ± MSE with n = 5. Control, SHAM, and TNEG animals receiving dilution solvent; LET: animals receiving letrozole at a dose of 10 mg/Kg BW/day; ASP: animals receiving aspirin at a dose of 3 mg/Kg BW/day; GPC: animals treated with aqueous extract of G. pictum at doses of 50 and 275 mg/Kg BW/day; GPM: animals treated with methanol extract of G. pictum at doses of 50 and 275 mg/Kg BW/day. # P < 0.05 corresponds to the degree of significance compared with the SHAM group * P < 0.05; ** P < 0.01 corresponds to the degree of significance compared with the TNEG group. PF, primary follicle; SF, secondary follicle; AF, antral follicle; GF, Graafian follicle; LC, corpora lutea. The results for some fertility parameters are showed in Table 2 . These results showed that the fertility index and pregnancy rate were significantly reduced in animals from the TNEG group compared with the SHAM group. However, an increase in these parameters was observed at all doses with all treatments compared to the TNEG group. This was approximately twice the rate observed in the TNEG group.

The female Wistar rats, aged 8–10 weeks at the start of experimentation and weighing around 140–160 g, used in the experiment were bred in the animal house of the Laboratory of Animal Physiology, Faculty of Science, University of Yaoundé I (Yaoundé, Cameroun). In groups of five, they were housed in plastic cages and were maintained at room temperature with adequate aeration under a natural light:darkness cycle. They had free access to drinking water and a soy-free rat diet ad libitum . All the treatment of rats and experiments were carried out according the guidelines and procedures of animal bioethics of the Cameroon Institutional Ethics Committee (CEE Council 86/609), which adopted all procedures established by the European Union on Animal Protection for scientific purposes. Efforts were made to minimize animal suffering and pain. G . pictum Griff is an herbaceous ‘caricature plant’ in the Acanthaceae family. This plant was collected in EKOUMDOUM (Mbankomo district, Mefou and Akono department, Central region of Cameroon), in the month of 2022 July. The botanical sample used was identified and authenticated by Dr TCHIENGEU Barthélémy, botanist at the National Herbarium of Cameroon (HNC) in Yaoundé, by comparison with sample No. 66900HNC. For this study, aqueous and methanolic extracts of G . pictum were used. Five hundred and ninety-two grams of dry G . pictum powder (whole plant) were boiled in 10 L of tap water for 30 min, then filtered and oven-dried at 45°C with a Memmert oven (southern Germany). Final extract of 20 g was obtained, giving a percent yield of 3.3%. For methanol extraction, 2 Kg of dry powder was macerated in 5 L of methanol for 48 h at room temperature. Using a rotary evaporator from Heidolph (Germany), 25 g of extract was obtained, for a 1.25% yield. Estradiol valerate (Progynova® 2 mg) was purchased from Delpharm (France); letrozole (2.5 mg) from Denk Pharma (Germany), aspirin or acetylsalicylic acid (Protect® 100 mg/kg) from Bayer Healthcare (France), and oxytocin (one ampoule containing 10 IU/mL) was supplied by LDI International N.V (Belgium). In women with oligoanovulation problems, letrozole not only promotes ovulation but also controls ovarian hyperstimulation ( Teede et al. 2018 ). The choice of 10 mg/kg letrozole dose in this experiment was based on data from previous studies ( Mvondo et al. 2017 ). According to the work of Pereira et al. (2015) , the 3 mg/kg dose of aspirin can reduce uterine weight and implant volume in endometriotic animals. The doses of 50 and 275 mg/kg of G. pictum aqueous and methanol extracts were taken from the work of Ketcha et al. (2016) . In order to develop more cost-effective procedures (reduction of experimental time and materials, risk of graft rejection), a slight modification of commonly used autografting techniques was used in this study. The animals were given 0.5 mg/kg estradiol valerate (estrogen) orally for 3 days ( Chen et al. 2015 , Mvondo et al. 2017 ). The aim was to create a hormonally favorable environment for implantation of endometriotic lesions, to reduce inter-subject variability, and to increase the reproducibility of endometriosis. Estrogen promotes vascularization and implants adhesion, stimulates endometrial proliferation as observed in estrus, and makes the grafted endometrial tissue more responsive. After estrogen administration, only female rats in estrus were retained for the continuation of the experiment, while those that were not in estrus were excluded. Estrus was confirmed by the presence of cornified epithelial cells (cells with irregular shape without nucleus) in vaginal smear analysis. Vaginal samples were taken and observed under the microscope as follows. Each vaginal smear was collected using a micropipette with a clean tip containing 10 μL of 0.9% NaCl solution. Subsequent to collection, the sample was expelled on a glass slide and air-dried. The slides were then observed under a microscope. This enabled the identification of the estrus phase of each animal. Ninety-day-old female Wistar rats were anaesthetized with diazepam (10 mg/kg) and ketamine (50 mg/kg) by intraperitoneal injection. Once asleep, the animals were placed in the dorsal recumbent position, then an abdominal midline incision (30 mm long) was made, and both uterine horns were exposed. The left uterine horn was incised longitudinally for 10 mm after removal of the ovary, which remained attached to the surrounding fat. This size corresponded to the tissue segment used for auto-transplantation. Without being completely excised over its width (about 2 mm left to maintain the blood supply), this fragment of the uterus was transplanted to the parietal peritoneum (internal abdominal wall) and ligated with silk thread (non-absorbable 2–0 polypropylene). Then, to prevent desiccation and reduce adhesion formation, 2 mL of saline solution (NaCl 9%) was administered into the abdominal cavity ( ip administration) after closure with the same thread, and the procedure was repeated the next day. Among these rats, we had a SHAM group ( n = 10) that underwent a white surgery with a simple suture procedure on the parietal peritoneum without any endometrial tissue being grafted. Subsequently, to prevent infection, 400.000 units of penicillin were injected intramuscularly daily at various sites for 7 days. Twenty-eight days later, these animals underwent laparotomy to verify the success of induction and to free the implanted fragment from the uterine horn to which it had partially attached. Animals with an implant (ectopic uterine tissue) were retained. Parameters such as implant length, width, and height were recorded to calculate the volume of endometriotic tissue before treatment. This was calculated using the following ellipsoid formula: Volume (mm 3 ) = 0.52 × width × length × height Next, the abdominal cavity was closed and irrigated with 2 mL of saline solution as before. Finally, the animals received penicillin as previously described. Fourteen weeks after the second laparotomy, the animals were divided into nine groups of ten animals each ( n = 10): a normal, SHAM, and negative control batch receiving 10 mL/kg distilled water (diluting solvent). Two positive control groups were included, one receiving aspirin at a dose of 3 mg/kg and one receiving letrozole at a dose of 10 mg/kg. The test groups received per os aqueous (6 and 7) and methanol (8 and 9) extracts at doses of 50 and 275 mg/kg body weight (b.w.), respectively, for 7 days. During this treatment period, estradiol valerate (0.5 mg/kg) was administered by gavage to all groups 6 h after administration of the vehicle, aqueous, and methanolic extracts of G . pictum . On the last day of treatment, to reproduce the intensity and frequency of uterine contractions in animals as observed during dysmenorrhea in humans, and to reduce animal stress and procedural variability, we chose a single, well-validated endpoint of abdominal writhing to assess pain. The animals were then weighed and subjected to a 30 min pain evaluation test. For this test, the animals received oxytocin by intraperitoneal injection at a dose of 2 IU per rat 1 h after E2V administration. Three persons were present to record parameters such as the number and latency time of writhing (abdominal contractions) for each animal. After writhing pain behavior, five animals of each treated group were immediately sacrificed, and blood, peritoneal fluid, vagina, uterus, mammary gland, and ovaries were subsequently removed. The uterus and right ovary were immediately weighed using a sensitive balance. The uterine implant (0.2 mg) was homogenized and centrifuged (1006.2 g at 5°C for 15 min) and stored at −20°C for later determination of biochemical parameters (Glutathione (GSH), Malondialdehyde (MDA), superoxide dismutase (SOD), nitrites). Peritoneal fluid was used to determine the levels of pro-inflammatory cytokines (IL-6 and TNF-α) and neovascularization (VEGF). Implants were also collected and measured (length, width, and height). Implants and other organs collected were fixed in 10% formalin for histological analysis. The remaining animals were mated with males of proven fertility to assess reproductive or fertility parameters (fertility index and pregnancy rate). They were calculated as follows: fertility index = (number of pregnant/number of mated) × 100, and pregnancy rate = (number of females with viable fetuses at birth/total number of gestational females) × 100 ( Awounfack et al. 2018 ). The latency time of writhing corresponded to the time between the application of the stimulus and the reaction it triggered in the rat. It was expressed in minutes. The writhing frequency corresponded to the ratio between the number of torsions and the duration of the experiment in the rats, multiplied by 100. It was expressed as a percentage: F = (number of writhing/writhing time (second)) × 100 The quantities of reduced glutathione (GSH), superoxide dismutase (SOD) activity, malondialdehyde (MDA), and nitrite were determined according to the protocols indicated by the manufacturers ( Wilbur et al. 1949 , Ellman 1959 , Misra & Fridovish 1972 , Slack 1987 ). Concentrations of cytokines, including TNF-α, IL-6, and VEGF, were determined in rat peritoneal fluid using the ELISA method, according to the manufacturer’s instructions. After fixation of the different organs in 10% formalin, the histological study techniques used were those described by Cannet (2006) . Microscopic analysis of the sections was then carried out using an Axioskop 40 microscope connected to a computer, where the images were transferred, edited, and analyzed using MRGrab 1.0 and Axio Vision 3.1 software, all supplied by ZEISS (Germany). The size of the uterine epithelium was measured and expressed in μm on the photomicrographs using the same software. The follicular population was studied by observing the number and size of follicles. Follicle identification was based on the detection of a nucleus and the type of epithelial cells surrounding it. Follicles were considered primary if they consisted of oocytes surrounded by a single layer of cuboidal follicular cells; those with more than one layer of follicular cells were pre-antral (secondary) follicles; antral follicles showed an antrum of follicular fluid, while Degraaf follicles were those with a single large antrum of follicular fluid with an eccentric oocyte. Ruptured follicles with enlarged follicular cells and a blood-filled cavity were considered to be corpora lutea. Data were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Dunnett’s post-test was used to determine the significance of the difference between negative control animals and treated animals. The significance of the difference between the SHAM group, control, and negative control animals was determined by the non-parametric Mann–Whitney U test. The ‘p’ probability values were used to determine the degree of significance. It was set at P < 0.05.

The aim of this study was to determine the anti-inflammatory and fertilizing properties of aqueous and methanolic extracts of G . pictum in an experimental rat model of endometriosis. The results showed that aqueous and methanolic extracts of G. pictum were able to stop the proliferation of endometriotic implants, including reducing implant volume, inhibiting the pain process induced by oxytocin in endometriosis, inhibiting oxidative stress and inflammation, and promoting fertility in rats with endometriosis. Aqueous and methanolic extracts of G. pictum could therefore be an alternative for women with endometriosis who wish to have children, but further research is needed.

G . pictum is an herbaceous plant with diverse biological effects, including antimicrobial, antioxidant, anti-inflammatory, analgesic, immunomodulatory, and estrogenic activities ( Ketcha et al. 2016 , Rahmah 2018 , Riwanto et al. 2020 , Makkiyah et al. 2021 ). Its phytochemical content includes flavonoids (rutin, hyperoside, and quercetin), steroids, glycosides, tannins, saponins, and alkaloids ( Aulia et al. 2018 , Makkiyah et al. 2021 ). This plant is used to treat female reproductive disorders in Cameroon ( Ketcha et al. 2016 ). In order to find an alternative treatment for inflammatory and painful conditions that can lead to female infertility, this work was designed to evaluate the effects of G . pictum extracts on a female Wistar rat model of induced endometriosis. The model used in this study is an adaptation of the model developed by Vernon & Wilson (1985) ( Mvondo et al. 2017 , Ilhan et al. 2019 ). Induction of endometriosis in rats followed by injection of oxytocin at a dose of 2 IU/rat after gavage of 0.5 mg/kg estradiol valerate (E2V) was characterized by a significant decrease in writhing latency and frequency, in serum levels of reduced GSH and SOD, followed by an increase in endometrial implant volume, in implant levels of malondialdehyde (MDA, a marker of lipid peroxidation) and nitrites, and in pro-inflammatory cytokines (IL-6, TNF-α and VEGF). These results confirm the success and establishment of endometriosis in these female Wistar rats. However, there was no development of endometriotic lesions in animals from the normal control and SHAM groups (animals with the same surgical procedure as the treated animal, but without the uterine implant). Painful menstruation or dysmenorrhea, dyspareunia, and infertility are some of the clinical signs and symptoms observed in women with endometriosis ( Meuleman et al. 2009 , Castro et al. 2021 , Chen et al. 2023 ). During menstruation, painful cramps felt in the lower abdomen and radiating to the back or thighs are called dysmenorrhea or menstrual cramps ( Koninckx et al. 2017 ). This pain is caused by high levels of prostaglandins (hormone-like substances produced by the uterus), which can increase uterine tone and contractions via G-protein-coupled receptors ( Narumiya & FitzGerald 2001 ). In rats, an intraperitoneal injection of 2 IU of oxytocin 30 min after the last administration of E2V is used to assess pelvic pain characterized by abdominal writhing ( Mvondo et al. 2017 ). Oxytocin binding to its G protein-coupled receptor stimulates the release of calcium ions, which bind to calmodulin and induce uterine contractions via the Gq/phospholipase C (PLC)/inositol 1,4,5-triphosphate (InsP3) signaling pathway ( Gimpl & Fahrenholz 2001 , Smith 2007 ). Similarly, oxytocin stimulates prostaglandin secretion via the MAP kinase (MAPK) signaling pathway at its site of action. This secretion is involved in the mechanism of myometrial contraction and causes vasoconstriction of small endometrial blood vessels, resulting in tissue ischemia, endometrial disintegration, bleeding, and pain ( Rosenwaks & Seegar-Jones 1980 , Vrachnis et al. 2011 ). The presence of endometrial tissue outside the uterus cavity (endometriosis) is thought to be the cause of more severe dysmenorrhea. The endometriotic cells are under the influence of estrogens and react cyclically with bleeding at the time of menstruation ( Lachat et al. 2013 ). The results obtained in this work showed a significant increase in the writhing frequency and a decrease in the writhing latency in the negative control group (TNEG) compared to the SHAM group ( P < 0.001). However, this variation was corrected by the treatments. A significant increase in the writhing latency (latency to first writhing) was observed in the aspirin group ( P < 0.01) and in animals treated with aqueous (275 mg/Kg BW) and methanolic (all doses) extracts of G. pictum ( P < 0.001). In addition, the reduction in the frequency of abdominal writhes reduced significantly with the methanolic extract at a dose of 50 mg/Kg BW ( P < 0.001) and in the letrozole ( P < 0.05) and aspirin ( P < 0.01) groups. These results suggest that letrozole, aspirin, and G. pictum extracts inhibit the estradiol-mediated signaling pathway, leading to a reduction in dysmenorrhea-like model parameters ( Mvondo et al. 2017 ). Studies have also shown that frequent use of non-steroidal anti-inflammatory drugs improves dysmenorrhea in 27–35% of cases ( Youngster et al. 2013 ). According to the literature, plants containing natural substances of elements such as alkaloids, flavonoids, steroids, and tannins have strong analgesic activity ( Tadiwos et al. 2017 ). In addition, some compounds found in G. pictum (saponins, alkaloids, and flavonoids such as quercetin) are thought to be involved in inhibiting the synthesis and release of various inflammatory mediators and suppressing the sensitivity of pain nociceptors ( Tadiwos et al. 2017 , Aulia et al. 2018 , Hutagalung et al. 2019 , Makkiyah et al. 2021 ). These observations confirm those of Makkiyah et al. (2021) on the analgesic activity of G. pictum leaves, which showed that the β-sitosterol present in this plant inhibits pain. Studies have also shown that G. pictum has the ability to inhibit cyclooxygenase-2, known as the enzyme that catalyzes the conversion of arachidonic acid to prostaglandin H2, the committed step in the formation of prostaglandins ( Wang & Dubois 2006 , Riwanto et al. 2020 ). Consequently, prostaglandins are involved in proliferation, invasion, migration, immunomodulation, and endometriosis-related pain. In fact, any substance with the ability to reduce the number of writhing/contortions possesses analgesic activity because it inhibits prostaglandin synthesis and release by blocking peripheral pain transmission ( Tadiwos et al. 2017 ). The imbalance between free radicals (reactive oxygen and nitrogen species) and antioxidants is at the root of oxidative stress. The binding of these unstable molecules to various cellular structures leads to cellular damage and disease ( Baboo et al. 2019 ). Numerous studies have shown increased oxidative stress in serum, peritoneal fluid, follicular fluid, ovarian cortex, and endometrial tissue of women with endometriosis ( Gennaro et al. 2017 , Zong et al. 2022 ). Oxidative stress plays an active role in the initiation, maintenance, and progression of endometriosis ( Turgut et al. 2013 ). In this study, a decrease ( P < 0.05) in antioxidant enzymes (GSH) and an increase in NO ( P < 0.05) were observed in the implant homogenate serum of animals in the TNEG group compared to the SHAM group. Although not significant, SOD and MDA levels showed a slight decrease (19.82%) and increase (44.70%), respectively, in the TNEG group compared to the SHAM group. In addition, these small variations are consistent with a significant decrease and increase in other oxidative biomarkers, as observed for GSH and NO levels, respectively, in this group. Although some of these variations did not reach significant levels, they may indicate a possible early stage of oxidative imbalance or a notable decline in antioxidant defenses. Further research is needed to determine whether these non-significant variations are transient or indicate a persistent antioxidant deficiency in endometriosis. All these results would be partly due to the local inflammatory state, which has the capacity to increase oxidative stress ( Alexandre et al. 2006 ). Indeed, activated endometrial tissue and macrophages (found in the peritoneal fluid) release pro-oxidant and pro-inflammatory factors involved in the formation of harmful reactive oxygen species ( Langendonckt et al. 2002 , Gennaro et al. 2017 ). The increased oxidative stress observed in the present study coincided with an increase in pro-inflammatory cytokines. There was a significant ( P < 0.001) increase in IL-6, TNF-α, and VEGF levels in TNEG animals compared to SHAM animals. This supports the involvement of pro-inflammatory and angiogenic factors in the proliferation and inflammation associated with the development and maintenance of endometriosis. IL-6, TNF-α, and VEGF are important mediators involved in the maintenance of endometriosis ( Griffith et al. 2010 , Ilhan et al. 2019 ). Binding of IL-6 and TNF-α to their membrane receptors accelerates inflammation and invasion at endometriotic sites via several signaling pathways such as phosphoinositide 3-kinase (PI3K), MAP kinase (MAPK), c-Jun N-terminal (JNK), and P38 ( Grung et al. 2008 , Cho et al. 2018 ). VEGF predominantly promotes the development of new blood vessels, growth, and inflammation of endometriotic foci. This result shows that angiogenesis occurs in the context of our work. Consistent with the literature, the endometriosis model used in this work is characterized by the presence of high levels of pro-inflammatory and pro-angiogenic substances in the peritoneal fluid of rodents ( Liang et al. 2019 , Castro et al. 2021 ). After a 7-day treatment period, aspirin (ASP) at 3 mg/Kg and G. pictum aqueous extract at 257 mg/Kg BW, induced a significant increase ( P < 0.001) in GSH levels and in serum SOD levels ( P < 0.05) at 50 and 257 mg/Kg BW with G pictum extracts, respectively. MDA was reduced ( P < 0.05) at the dose of 50 mg/Kg BW with G. pictum methanolic extract, in contrast to NO, which was reduced ( P < 0.001) at all doses as well as in animals in the aspirin (ASP) and letrozole (10 mg/Kg BW) groups. These results support the idea that aqueous and methanolic extracts of G. pictum have antioxidant activity. SOD is an antioxidant enzyme that repairs and reduces cellular damage caused by superoxide in the inflammatory process ( Hashempur et al. 2017 ). According to the work of Riwanto et al. (2020) , G. pictum can increase serum SOD levels. Indeed, through its phytochemical composition rich in flavonoids, tannins, saponins, steroids, resins, anthraquinones, coumarins, glucosides, and vitamin C ( Kumar & Pandey 2013 , Jiangseubchatveera et al. 2017 ), G. pictum possesses strong antioxidant or anti-free radical, anti-proliferative, and anti-inflammatory potential ( Jiangseubchatveera et al. 2017 , Kusumaningsih et al. 2018 , Rahmah 2018 , Riwanto et al. 2020 ). Phytoestrogenic compounds (flavonoids) have been reported to exert direct antioxidant effects by preventing intracellular hydrogen peroxide accumulation, inducing a decrease in the production of reactive oxygen species (ROS), limiting lipid peroxidation, protecting against oxidative stress, and reducing hydrogen peroxide levels ( Jakob et al. 2011 ). After the pain test, the reduction in oxidative stress observed with the treatment coincided with a reduction in pro-inflammatory cytokines. All the treatments induced a significant reduction ( P < 0.001) in the levels of IL-6, TNF-α, and VEGF in the peritoneal fluid, except for 50 mg/Kg BW in the GPC group. Letrozole is an aromatase inhibitor, suggesting that it promotes the reduction of estrogen levels and consequently reduces vascular endothelial cell proliferation and growth ( Bilotas et al. 2010 , Zhao et al. 2015 ). Aspirin is a non-steroidal anti-inflammatory drug that inhibits cyclooxygenase-2, which is responsible for the production of prostaglandin 2 and estrogens. The latter are known to increase the proliferation and expression of VEGF, which ensures the survival of endometrial stem cells outside the uterus ( Morimoto et al. 2005 , Melincovici et al. 2018 , Zhen–Zhen et al. 2019 ). According to the results of this work, G. pictum extracts possess antiproliferative and anti-angiogenic activities. These effects can be attributed to its previously mentioned phytochemical constituents ( Aulia et al. 2018 , Makkiyah et al. 2021 ), which possess strong immunomodulatory and anti-inflammatory potential ( Rahmah 2018 , Riwanto et al. 2020 ). Endometriosis, a major cause of infertility, is associated with frequent anovulation with destruction of corpus luteum function, and uterine receptivity dysfunction, which has an anti-implantation effect ( Sanchez et al. 2017 , Cheng et al. 2022 ). Oxidative stress negatively affects fertility by reducing ovarian reserve, and the inflammatory process is known to lead to implantation failure ( Wu et al. 2022 , Chen et al. 2023 ). In this study, the reduction in fertility due to endometriosis was characterized by a significant ( P < 0.05) decrease in the number of corpora lutea in the TNEG group compared to SHAM. The corpus luteum is known to play a crucial role in fertility as it is directly linked to the number of ovulated eggs and produces progesterone (hormone that prepares the endometrium for implantation and supports early pregnancy). Inadequate progesterone production can then lower the fertility index by causing implantation failure or early pregnancy loss ( Awounfack et al. 2018 ). After treatment, the aqueous and methanolic extracts of G. pictum significantly increased the relative mass of the ovaries ( P < 0.01) at all the tested doses, and the number of mature follicles (antral and Graafian follicles ( P < 0.05)) and corpus luteum ( P < 0.01) with GPC 50 and GPM at all doses. This result suggests that G. pictum extracts have the ability to stimulate follicular growth and maturation, as well as ovulation. This plant would therefore be an alternative for maintaining fertility in cases of endometriosis. The study of some fertility parameters in this work supports this idea. An increase in the percentage of the fertility index was observed in endometriotic rats after 7 days of treatment with aqueous and methanolic extracts of G. pictum . The antioxidant and anti-inflammatory activity of the plant is thought to not only normalize ovulation, but also promote embryo implantation and enhance fertility. Especially, the methanolic extract at the lowest dose of 50 mg/Kg BW had an effect on most of the parameters evaluated. Although an aqueous extract of G. pictum has previously shown weak estrogenic effects in a postmenopausal-like model of ovariectomized Wistar rats ( Ketcha et al. 2016 ), as a phytoestrogen, it did not enhance the estrogen-dependent development of endometriosis in this study. Estrogen is known to exacerbate endometriosis; however, not all phytoestrogens have the same potency as endogenous estrogen, and their effects may vary depending on the individual, the specific phytoestrogen compound ( Safe & Gaido 1998 , Shanle & Xu 2010 , Canivenc-Lavier & Bennetau-Pelissero 2023 ), the presence or absence of endogenous estrogens ( Andersen 2000 ), and the types of receptors activated and their distribution ( Andersen 2000 , Morito et al. 2001 , Paterni et al. 2014 ). Furthermore, even if estrogens promote the progression of endometriosis mainly by binding to different estrogen receptors (ERα, ERβ, and the estrogen receptor coupled to the membrane-associated protein G-GPER), the effect of estrogens on endometriosis is closely related to the types of these receptors and their distribution. A higher expression profile of ERβ than ERα has been proposed as an important background for endometriosis ( Izawa et al. 2016 ). Lin et al. (2017) observed that ERα levels are lower than ERβ in endometriotic stromal cells and tissues compared to eutopic endometrium. In addition, most of the undesirable proliferative effects of estrogens involve activation of ERα, such as cell growth. Then, in the presence of endogenous estrogens, as observed in this study, G. pictum can be considered as an ERβ-selective agonist, known for its therapeutic use in some types of cancer and several other inflammatory diseases ( Paterni et al. 2014 ). However, further studies are needed to investigate the effects of G. pictum on different estrogen receptors, and also to look at serum progesterone levels as a proxy for corpus luteum activity, and finally to relate this to fertility outcomes such as embryo viability or litter size in animals. All of this could help to better understand the mechanism of action of these extracts on endometriosis, as well as the potential long-term efficacy of this plant as a fertility treatment in endometriotic rats. In addition, although fibrosis was not assessed in our study, previous research has demonstrated an association between increased fibrosis in human ectopic endometrial lesions and a progressive decrease in estrogen signaling, particularly in deep endometriosis ( Yuan et al. 2023 , Nie et al. 2025 ). These findings suggest that fibrotic remodeling may modulate hormonal responsiveness, thereby contributing to the limited efficacy of estrogen-targeted therapies. Incorporating fibrosis quantification into our future work would be valuable for exploring this interaction further, for example, evaluating collagen deposition, fibroblast/myofibroblast presence, or fibrotic markers in ectopic lesions. The study of plant toxicity is essential to protect health, advance scientific research, and ensure environmental and agricultural safety. Although extracts of G. pictum are considered by the general public to be an ornamental plant, edible and harmless to the body, and given its growing interest in the scientific community in recent years, an in-depth study of the toxicity (acute and sub-acute) of this plant is urgently required in order to promote its beneficial effects in the treatment of endometriosis.

Endometriosis is an inflammatory gynecological disorder characterized by the presence and growth of fragments of endometrial tissue on a surface outside the uterine cavity ( Organisation mondiale de la santé (OMS) 2018 , Vermeulen et al. 2021 , Tejada et al. 2023 ). The ectopic lesions are commonly found on the pelvic organs and peritoneum, but can also be observed on the bowel, pleura, pericardium, ovaries, bladder, kidneys, lungs, abdominal skin, and brain ( Bulun et al. 2019 , Vermeulen et al. 2021 , Bashir et al. 2023 ). However, accurate and widespread determination of its prevalence, as well as its incidence, is not evident, as very few women are diagnosed ( Borghese et al. 2018 ). In addition, depending on the age of patients, body weight, ethnicity, country, time of diagnosis, and type of endometriosis ( Mick et al. 2025 ), diagnosis is often delayed by 2–13 years after symptom onset ( Ballard et al. 2006 , Harder et al. 2024 , De Corte et al. 2025 ), worsening the patient’s condition ( Surrey et al. 2020 ). This progressive, estrogen-dependent disorder affects approximately 10% of the female population of reproductive age, and it is estimated that about 190 million women and girls globally are living with this disease ( Zondervan et al. 2020 , Horne & Missmer 2022 , Tejada et al. 2023 ). Between 49 and 75% of adolescents with pelvic pain and up to 50% of infertile women worldwide are also affected ( Tejada et al. 2023 ). The prevalence is much higher in women with chronic pelvic pain ( Shafrir et al. 2018 ) and causes difficulty conceiving in 50% of women ( Fadhlaoui et al. 2014 , Zondervan et al. 2020 ). The incidence of scar endometriosis has increased due to high rates of cesarean section ( Andolf et al. 2013 , Monist et al. 2019 ). Globally, the incidence is regionally increased, and the disease burden may be underestimated in some low- and middle-income regions due to limited data or lack of gold standard diagnosis ( Li et al. 2025 ). Chronic pelvic pain and/or infertility are the most common clinical symptoms ( Tejada et al. 2023 ) observed in 30–50% of women with endometriosis ( Taylor et al. 2021 , Bashir et al. 2023 ) and are associated with psychological disorders such as depression (around 86% in women with endometriosis-associated chronic pelvic pain), anxiety, stress, and sadness ( Chen et al. 2016 , Bashir et al. 2023 ). All these symptoms have a negative impact on the quality of life of women with endometriosis ( Simoens et al. 2012 , Falcone & Flyckt 2018 ), as well as increased socioeconomic costs in terms of healthcare expenditure ( Soliman et al. 2018 ). Other symptoms of endometriosis include dysmenorrhea, menorrhagia, dyspareunia, dysuria, bowel pain (rectal and appendicular), and gastroesophageal reflux disorders ( Davis & Goldberg 2017 , Bulun et al. 2019 , Bashir et al. 2023 ). Due to the common innervation of the reproductive tract, colon, and bladder, endometriosis-associated comorbidities include overactive bladder syndrome and irritable bowel syndrome ( Maddern et al. 2020 , Bashir et al. 2023 ). Endometriosis is a multifactorial condition with an unknown cause resulting from the combination of numerous genetic, environmental, immunological, and menstrual factors ( Fauconnier et al. 2018 ). Some of the factors contributing to the chronic pelvic pain observed in this condition include inflammation associated with endometriotic lesions and the peritoneum, activation of peripheral nerve endings, central sensitization ( Manconi et al. 2018 , Zheng et al. 2019 , Bashir et al. 2023 ), and the immune system ( Kralickova & Vetvicka 2015 ). In the immunological aspect of endometriosis, 95% of activated macrophages found in the peritoneal fluid have impaired phagocytic function ( Khoufache et al. 2012 , Akoum & Khoufache 2015 ). These macrophages are responsible for the exacerbated production of pro-inflammatory cytokines (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8), growth factors (vascular endothelial growth factor (VEGF)), and pro-oxidants, which are responsible for the maintenance and progression of endometriosis ( Ahn et al. 2015 , Wu et al. 2017 , García-Gómez et al. 2020 ). These inflammatory cytokines are at the origin of the action of cyclooxygenase 2 (COX2), an enzyme that increases prostaglandin levels, which not only induce pain but also promote aromatase activity, thereby increasing estrogen secretion ( Hemani et al. 2018 , Xu et al. 2025 ). In endometriosis, a positive feedback between inflammatory mediators and estrogen (E2) has been observed ( Attar et al. 2009 ). According to studies, estrogens are involved in the maintenance and progression of endometriosis by stimulating COX2 activation, which increases PGE 2 production, forming a vicious circle ( Galvankar et al. 2017 , García-Gómez et al. 2020 ). However, recent studies have challenged the traditional view of endometriosis as a disease that is dependent on estrogen. In particular, it has been demonstrated that estrogen signaling progressively diminishes as fibrotic tissue increases, particularly in deep endometriosis, which exhibits the highest fibrotic content and the lowest responsiveness to hormonal treatments. This inverse relationship between fibrosis and estrogen receptor expression suggests that advanced lesions may become less sensitive to hormones, thereby contributing to therapeutic resistance ( Nie et al. 2025 ). Current pharmacological treatments, such as steroidal contraceptives, nonsteroidal, anti-inflammatory drugs, aromatase inhibitors, GnRH analogs, and analgesics, are commonly used, and in some cases, surgery is necessary ( Schwartz et al. 2020 ). These treatments aim to inhibit the production of estrogen, thereby alleviating pain caused by endometriosis. Unfortunately, they are ineffective for many women and reduce estrogen levels. However, the drop in estrogen levels adversely influences the ovarian follicular reserve, preventing the proliferation of the uterine epithelium, resulting in failure of embryo implantation ( Cheng et al. 2022 ), leaving women unable to conceive during treatment. In addition, these treatments have harmful side effects such as bone density loss, hot flushes, mood changes, thromboembolism, nausea, weight gain, vaginal dryness and atrophy, hirsutism, renal and liver toxicity, and an increased risk of estrogen-dependent cancers ( Pino 2017 , Horne & Saunders 2019 , Eva & Bill 2024 ), so alternative solutions need to be explored. Graptophyllum pictum ( G. pictum ) (L.) Griff, a member of the Acanthaceae family, is a tropical shrub commonly known as the caricature plant. It is traditionally used for its phytoestrogenic effects, and antioxidant, anti-inflammatory, and anxiolytic properties, and emollient, laxative, and diuretic activities ( Makkiyah et al. 2021 ). G. pictum is a plant used in traditional medicine to treat various ailments such as sores, swellings, and wounds; hemorrhoids, tonsillitis, ulcers, abscesses (including breast abscess), breast engorgement, constipation, urinary tract infections, rheumatism, scabies, hepatomegaly, ear diseases, pain relief, and menstrual problems ( Perry 1980 , Makkiyah et al. 2021 ). In traditional medicine in Cameroon, this plant is used to treat reproductive disorders in women ( Ketcha et al. 2016 ). Previous studies have shown that G. pictum exerts weak phytoestrogenic effects, potentially acting as an estrogen receptor modulator ( Ketcha et al. 2016 ). It promotes the expression of superoxide dismutase (SOD) and can inhibit the action of COX-2, thereby preventing the stimulation of prostaglandin ( Riwanto et al. 2020 ). Prostaglandin is considered to be a key player in pelvic pain, including endometriosis. The treatment of endometriosis through pain inhibition leads to an improvement in the psychological state and quality of life of women suffering from endometriosis ( Bulun et al. 2019 , Zong et al. 2022 ). Based on this previous result, G. pictum could be a therapeutic alternative for endometriosis patients who wish to conceive.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

SD and DN contributed to the study subject proposal. MPA, SD, CFA, and DN contributed to the project design. MPA and CFA were responsible for the methodology. MPA and CFA contributed to interpretation and analysis of data. MPA and CFA contributed to writing draft preparation. CFA contributed to drawing the graphical abstract. SD and DN contributed to writing, review, and editing. All authors read and approved the final manuscript.