Enoghase Itua Erasmus

PHARMACOLOGICAL AND UTEROTONIC EFFECTS OF AZADIRACHTA INDICA SEED EXTRACT AND OIL ON ISOLATED MOUSE UTERUS ABSTRACT This study evaluated the uterotonic and pharmacological effects of Azadirachta indica (neem) seed extract and oil on isolated uterine tissues of both pregnant and non-pregnant mice, using in vitro organ bath experiments and in vivo models. Administration of the methanolic seed extract (MSA) at low doses (0.1 mg/mL) significantly increased the frequency of spontaneous uterine contractions (p < 0.01), while higher doses (1.0 mg/mL) caused a marked reduction in contraction amplitude (p < 0.001) in both pregnant and non-pregnant tissues. Oxytocin-induced contractions (11.540 nM) were significantly inhibited by MSA (p < 0.001), and similar inhibition was observed with KCl-induced contractions (80 mM) (p < 0.05). In antagonist studies, the extract’s inhibitory effect on spontaneous contractions was reduced in the presence of tetraethylammonium (TEA) and propranolol (PR), indicating the involvement of potassium channels and β-adrenergic receptors. Notably, MSA co-administered with NM and PR led to a significant increase in contraction frequency (p < 0.05), suggesting a complex interaction with adrenergic and ionic pathways. In vivo antifertility evaluation showed that animals treated with high doses of MSA (1000 mg/kg) exhibited significantly reduced uterine weights compared to control groups (p < 0.01). The group treated with estradiol (10 mg/kg) showed the lowest uterine weight overall. Histological examination revealed reduced uterine glandular development and morphological alterations in treated groups, supporting the antifertility potential of the extract. These findings demonstrate that Azadirachta indica seed extract and oil exhibit dose-dependent uterotonic and antifertility effects, likely mediated by modulation of intracellular calcium and adrenergic signaling. The study supports traditional claims of the plant’s uterine activity, though safety concerns regarding reproductive toxicity warrant further investigation. Keywords: Azadirachta indica, Uterotonic activity, Uterine contractility, Adrenergic signaling, Antifertility effect 1 Introduction The use of herbal medicines has become increasingly popular worldwide, especially among women seeking natural approaches to reproductive health. In both developing and developed countries, a significant proportion of the population relies on plant-based remedies for managing fertility, pregnancy, and childbirth. Uterotonic plants—those capable of stimulating uterine contractions—have been traditionally employed to induce or augment labor, manage postpartum hemorrhage, and serve as abortifacients. Despite their widespread use, the safety and efficacy of many of these botanicals remain inadequately studied, raising concerns about potential adverse outcomes such as fetal distress and increased rates of cesarean section (Gruber & Brien, 2011; Mabina et al., 1997). The World Health Organization estimates that up to 85% of women in some regions depend on traditional healthcare systems during pregnancy and postpartum periods. This reliance is often influenced by cultural practices, family recommendations, and limited access to conventional medical care (Abdul et al., 2014). However, the use of herbal uterotonics is not without risk, and their pharmacological properties, mechanisms of action, and safety profiles require rigorous scientific evaluation. Among the most prominent and widely used medicinal plants is Azadirachta indica A. Juss (Neem), renowned in Asia and Africa for its diverse therapeutic applications. Neem has been incorporated into traditional remedies for centuries, attributed to its rich content of bioactive constituents with purported antifertility, abortifacient, and uterotonic effects (Abdul et al., 2014). While several studies have explored the impact of neem on male and female fertility, there remains a notable scarcity of data regarding its direct influence on uterine contractility and the underlying mechanisms involved (Mariyam et al., 2015). Previous research has suggested that neem seed extract and oil may modulate smooth muscle activity, potentially through interactions with calcium signaling and adrenergic pathways, yet systematic pharmacological evidence is limited. Therefore, this study aims to evaluate the effects of Azadirachta indica seed extract and oil on isolated mouse uterine tissue, providing experimental insight into its uterotonic potential and laying the groundwork for future investigations into its safety and clinical relevance. Literature Review Origin, distribution, and taxonomy of Azadirachta indica (neem tree) Azadirachta indica, commonly known as the neem tree, is believed to have originated from the Indian subcontinent, especially in India and Bangladesh, although its exact origin is not fully known. The species grows widely across South and Southeast Asia, and has also spread to over 70 countries, including those in Africa, America, and Australia. There are two main types: Azadirachta indica and Azadirachta excelsa, the latter being native to parts of Indonesia and the Philippines. The neem tree is highly valued for its resilience, as it grows well in different climates, especially in dry and nutrient-poor soils, and can survive temperatures ranging from 0°C to 49°C (Sateesh, 1998; Hedge, 1995). The neem tree has been used for thousands of years in traditional Indian medicine (Ayurveda), and almost every part of the plant—leaves, seeds, bark, fruits, and roots—contains compounds that help treat infections and diseases. Its name in Sanskrit, arista, means "perfect and imperishable," highlighting its healing reputation. The neem tree is known to have antifungal, antibacterial, antiviral, and anti-inflammatory properties. It is also considered one of the most 2 promising trees in the world for natural pest control and environmental protection. Its economic and medicinal importance has made it one of the most researched trees globally (Brahmachari, 2004; Thakkar, 1997). Botanically, neem is a large tree that can live up to 200 years and starts producing fruit after about 3 to 5 years. A fully grown tree can reach 25 meters in height and produce around 50 kilograms of fruit annually. Its leaves are green and toothed, flowers are small and white, and the fruits are yellow when ripe, resembling olives. Although birds eat the fruits, they are toxic to mammals. Neem trees grow best in deep, well-drained soils and are capable of improving soil conditions, including reducing acidity through calcium accumulation (Girish & Shankara, 2008; Kumar & Gupta, 2002). Pharmacological actions of neem extract Neem extract possesses a wide range of pharmacological properties that make it highly valuable in traditional and modern medicine. One of its key components, gedunin, found in the seed extract and oil, has proven antifungal and antimalarial effects. Another compound, azadirachtin, not only acts as a strong insect antifeedant but also inhibits the development of malaria parasites. Additionally, mahmoodin, a deoxygedunin compound also extracted from neem seed oil, has shown moderate antibacterial activity against certain human pathogenic bacteria. The bark of the neem tree contains condensed tannins rich in bioactive compounds like gallic acid, gallocatechin, epicatechin, catechin, and epigallocatechin. Among these, gallic acid, catechin, and epicatechin have been identified as significant inhibitors of oxidative burst in human polymorphonuclear neutrophils (PMNs), a process linked to inflammation. This suggests neem’s potential in managing inflammatory conditions. Numerous studies confirm that the biological activity of neem extends to crude extracts and isolated fractions from almost every part of the plant, including its leaves, bark, roots, seeds, and oil, supporting its widespread use in herbal and pharmaceutical applications (Pathak, 2013; Brahmachari, 2004; Girish & Shankara, 2008; Biswas et al., 2002). Neem as a Uterotonic: Traditional and Experimental Perspectives Azadirachta indica A. Juss, commonly known as neem, is a fast-growing evergreen tree native to the Indian subcontinent but now widely distributed across tropical and subtropical regions of Africa, Asia, and beyond. Traditionally, neem has been revered in Ayurvedic and African folk medicine for its remarkable therapeutic versatility, with various parts of the plant—including leaves, bark, seeds, and oil—used to treat infections, inflammation, and reproductive disorders (Pathak, 2013). In the context of reproductive health, neem seed extract and oil have been employed for their antifertility, abortifacient, and uterotonic effects, particularly by traditional birth attendants to induce labor, manage postpartum bleeding, and as abortifacients in cases of unwanted pregnancy (Mariyam et al., 2015). These uses are deeply embedded in cultural practices, often passed down through generations and recommended by family members or local healers. Despite their popularity, the scientific validation of neem’s uterotonic properties remains limited, and the safety profile of such applications is not well established, especially considering the potential for adverse obstetric outcomes such as fetal distress and increased risk of cesarean section (Rico, 2016). Experimental studies in animal models have provided some support for the traditional claims regarding neem’s reproductive effects. Research has shown that neem oil and extracts can induce abortion, inhibit implantation, and alter hormonal profiles in rodents, suggesting a direct 3 influence on uterine physiology (de Souza et al., 2013). The uterotonic activity of neem is thought to be mediated by its diverse array of bioactive constituents, including terpenoids, flavonoids, and fatty acids, which may modulate smooth muscle contractility through pathways involving calcium signaling and adrenergic receptors—mechanisms similar to those of established uterotonic agents such as oxytocin and prostaglandins (Chakraborty et al., 2014). However, the specific molecular pathways, dose-response relationships, and comparative efficacy of seed extract versus oil are not fully understood. Furthermore, while some studies have indicated that neem can interact with uterine adrenergic and calcium channels, systematic pharmacological investigations remain scarce (Olubunmi et al., 2022). Thus, there is a clear need for rigorous experimental research to elucidate the pharmacodynamics, safety, and clinical potential of neem as a uterotonic agent, particularly given its widespread use in traditional medicine. Materials and Methods Animal Model Thirty five (35) adult female Swiss albino mice (20–30 g) both non-pregnant and pregnant (day 9 of gestation), were purchased from the Animal House Department of Pharmacology & Toxicology, University of Benin, Nigeria and housed under standard laboratory conditions. All protocols were approved by the institutional animal care and use committee. Preparation of Extract and Oil Neem seeds were collected, air-dried, and ground. The aqueous extract was prepared by maceration and filtration, while the oil was obtained by cold pressing. Both were stored at 4°C until use. Concentrations for in vitro and in vivo experiments were standardized based on preliminary phytochemical screening. Uterine Contractility Assays Isolated uterine strips were mounted in organ baths containing physiological saline solution at 37°C and aerated with 95% O₂/5% CO₂. Spontaneous, oxytocin-induced, and KCl-induced contractions were recorded using isometric force transducers. Cumulative concentrations of neem seed extract and oil were added, and changes in amplitude and frequency of contractions were measured. The role of receptor pathways was assessed using antagonists such as propranolol, tetraethylammonium (TEA), and N-methyl-D-glucamine (NM). In Vivo Implantation and Uterine Weight Pregnant mice received oral administration of extract or oil at 10, 100, and 1000 mg/kg for five days. On day 9 of gestation, implantation sites were counted and uterine weights measured. Data Analysis The mean frequency and amplitude were calculated from contractions occurring within 5 min of the phasic contractions using the GraphPad Prism, (version 7.1; GraphPad software Inc, San Diego, CA, USA). Results were obtained as control vs treatment responses. All data shown were expressed as mean ± standard error of mean (SEM) and ‘n’ represented the number of uterine tissues from different animals. Significance was evaluated using appropriate t-tests, and P values ≤ 0.05 were taken to represent minimum significance. Results Contractility Both neem seed extract and oil produced concentration-dependent effects on uterine contractility. In non-pregnant mice, low concentrations increased the frequency of spontaneous contractions, while higher concentrations inhibited amplitude. In pregnant mice, both extract and oil inhibited 4 the amplitude of contractions and, at higher concentrations, overcame the initial increase in frequency. The extract also significantly inhibited oxytocin- and KCl-induced contractions, with effects attenuated by propranolol, TEA, and NM, indicating involvement of adrenergic and potassium channels. The extract (MSA) caused a concentration-dependent inhibition of spontaneous uterine contractility (Fig. 1). Figure 1. Representative original tracing showing the effect of MSA on spontaneous uterine contraction in the non-pregnant uterus. 5 Figure 2. Concentration-response curves showing the effect of MSA on spontaneous uterine contraction in the non-pregnant mouse uterus. (A) MSA inhibited the amplitude of contractions; (B) An initial increase was observed in the frequency which was overcome at higher concentrations. n = 6 animals. Effect of Azadirachta indicaon spontaneous contractions in the pregnant uterus MSA caused a concentration-dependent inhibition of spontaneous contractions in the non-pregnant uterus (Fig. 3) Figure 3. Representative original tracing showing the effect of MSA on spontaneous contractions in the pregnant uterus. 6 Figure 4. Bar graphs showing the effect of MSA on the amplitude and frequency of spontaneous contraction in the pregnant uterus. (A) MSA caused an inhibition of the amplitude of contractions; (B) MSA caused an increase in the frequency of contractions which were overcome at higher concentrations of MSA. n= 6 animals. Effect of Azadirachta indica on oxytocin-induced uterine contraction in the non-pregnant uterus MSA was tested on OT-induced contractions in the non-pregnant uterus where it caused an overall inhibition in OT-induced contraction (Fig. 5). On analysis, MSA significantly (p<0.001) inhibited the amplitude of OT-induced contraction (Fig. 6A) while an increase in the frequency of OTinduced contraction was observed which was overcome as the concentration of MSA increased (Fig. 6B). Figure 5. Representative original tracings showing the effect of MSA on OT-induced uterine contraction in the non-pregnant uterus. 7 Figure 6. Bar graphs showing the effect of MSA on the amplitude and frequency of OT-induced uterine contraction in the non-pregnant uterus. (A) MSA inhibited the amplitude of OT-induced uterine contractions; (B) an increase in frequency was observed which was overcome at higher concentrations. n = 6 animals; ***p< 0.001 compared to OT in the absence of MSA. Effect of Azadirachta indica on high KCl-induced uterine contractions in the non-pregnant uterus MSA (0.1 mg/mL) was tested on high KCL (80 mM)-induced uterine contraction in the nonpregnant uterus (Fig. 7). MSA caused a significant inhibition (p<0.05) on KCL-induced contraction (Fig. 8). Figure 7. Representative original tracing showing the effect of MSA on KCl-induced uterine contraction in the non-pregnant uterus. 8 Figure 8. Bar graphs showing an analysis of the effect of MSA on KCL-induced contraction in the non-pregnant uterus. MSA clearly inhibited the amplitude of KCL-induced contraction. n = 6 animals; *p<0.05 compared to the KCl in the absence of MSA. Uterine Weight and Implantation Oral administration of neem extract and oil resulted in a significant reduction in uterine weight and number of implantation sites compared to controls. The highest dose group showed nearcomplete inhibition of implantation, while estradiol-treated animals had the lowest uterine weights. Table 1. Number of implantation sites in control and treated mice after administration of neem seed extract/oil and estradiol. Groups Number of animals without Total Number of % inhibition of implantation sites implantations implantation 0 8.83 ±- ±1.17 No inhibition 0 7.83 ±- ±- ±- Group 1 (No treatment) Group 2 (10% Tween 80) Group 3 (MSA 10 mg/kg) Group 4 (MSA 100 mg/kg) Group 5 (MSA 1000 mg/kg) Group 6 (10 mg/kg estradiol) n= 6 animals “The number of implantation sites was significantly reduced in treated groups compared to control (Table 1).” 9 Figure 18. Bar graphs showing the weight of uterus on Day 9 of gestation after treatment with MSA. The animals without treatment showed a higher uterine weight while the animals treated with MSA showed a lower uterine weight. Animals treated with estradiol (E2) showed the lowest uterine weights. n= 6 animals; **p<0.01 compared to the group with no treatment. A B D E C F 10 Plate 2: Effect of treated extracts group and control on the Uterus. A. Normal control: Sections of the uterus reveal numerous endometrial glands (long arrow) which appear simple and circular in forms embedded in connective tissue lamina propria. B. Control tween 80 F8: Sections of the uterus reveal visible endometrial glands (long arrow) in circular forms embedded in connective tissue lamina propria and visible dense mononuclear cells C. 10mg/kg extract F14: Sections of the uterus reveal visible endometrial glands with slightly increased mitotic activities and nuclear chromasia (long arrow) there is diffused mononuclear infiltrates in the connective tissue lamina propria. D. 100mg/kg extract F20: Sections of the uterus reveal endometrial glands with sub-nuclear vacuoles (long arrow). There is distortion connective tissue lamina propria with prominent mononuclear infiltrates. E. Estrogen F31: Estrogen uterus micrographs reveal prominent endometrial glands which appear simple in circular forms with visible conspicuous mitotic activity evident of proliferative changes (long arrow). A B D E C F Plate 8: showed the effect of treated extracts group and control on the Uterus A. Normal control of the uterus: Sections of the uterus reveal numerous endometrial glands (long arrow) which appear simple and circular in forms embedded in connective tissue lamina propria. 11 B. Control tween 80 F2: Sections of the uterus reveal visible endometrial glands (long arrow) in circular forms embedded in connective tissue lamina propria and visible dense mononuclear cells. C. 10mg/kg extract F16: Sections of the uterus reveal visible endometrial glands with slightly increased mitotic activities and nuclear chromasia (long arrow) there is diffused mononuclear infiltrates in the connective tissue lamina propria. D. 100mg/kg extract F24: Sections of the uterus reveal endometrial glands with sub-nuclear vacuoles (long arrow). There is distortion connective tissue lamina propria with prominent mononuclear infiltrates. E. 1000mg/kg extract F30: Sections of the uterus reveal visible endometrial glands with nuclear stratification and hyperchromasia. There is also increased mitotic activities (long arrow) embedded in connective tissue lamina propria (short arrow). F. Estrogen group F36: Estrogen uterus micrographs reveal prominent endometrial glands which appear simple in circular forms-with visible conspicuous rnitotic activity evident of proliferative changes (long arrow). Discussion The present study provides compelling evidence that Azadirachta indica (neem) seed extract and oil possess significant uterotonic and antifertility activities in mice. The extract and oil both produced a distinct biphasic effect on uterine contractility: at lower concentrations, there was an increase in the frequency of spontaneous uterine contractions, while at higher concentrations, a marked inhibition of contraction amplitude was observed (see Figures 1 and 2). This pattern suggests that neem can initially stimulate uterine activity, potentially facilitating labor, but at higher doses may suppress effective contractions, which could interfere with normal parturition. Such biphasic responses have been reported in other uterotonic herbs and are often attributed to the presence of multiple bioactive compounds acting on different cellular targets (Bafor and Sanni, 2017). In addition to the in vitro contractility findings, in vivo experiments demonstrated a significant reduction in uterine weight and the number of implantation sites in mice treated with neem extract and oil compared to controls (see Table 1 and Figure 3). These results support the traditional use of neem as an abortifacient and contraceptive agent in various cultures (de Souza et al., 2013). The antifertility effect is likely due to both direct inhibition of uterine muscle contractility and interference with implantation processes, as previously observed in rodent studies (Olubunmi et al., 2022). The gross morphological changes in the uterus, as shown in Plates 2 and 5, further corroborate the physiological impact of neem treatment on reproductive tissues. The observed biphasic contractile response may be explained by neem’s modulation of calcium influx and adrenergic pathways, as supported by mechanistic experiments in the companion article and by literature on uterine smooth muscle physiology (Wray, 2007; Sophia et al., 2020). At lower concentrations, neem constituents might enhance calcium entry or sensitize adrenergic receptors, increasing contractile frequency. At higher concentrations, however, the inhibition of amplitude suggests a blockade of calcium channels or antagonism of stimulatory pathways, resulting in reduced effective contractions. This dual action highlights the pharmacological complexity of neem and underscores the importance of dose in determining its uterotonic or tocolytic outcome. 12 While these findings validate traditional claims regarding neem’s use in fertility regulation, they also raise important safety considerations. The reduction in uterine weight and implantation rates points to potential risks if neem is used inadvertently during early pregnancy, as it may compromise uterine receptivity and embryo survival (de Souza et al., 2013). Furthermore, the variability in response depending on dose and physiological state suggests that unsupervised use of neem could lead to unpredictable reproductive outcomes, including incomplete abortion or uterine atony. Therefore, further research is needed to identify the specific active compounds responsible for these effects, clarify their mechanisms of action, and evaluate the long-term safety profile of neem preparations in reproductive health contexts (Olubunmi et al., 2022). Conclusion Azadirachta indica seed extract and oil possess notable uterotonic and antifertility properties, as evidenced by their effects on uterine contractility, implantation, and uterine weight in mice. 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