Hrev_master Healthcare in Low-resource Settings 2025; volume 13:14021 The effect of 96% ethanol extract of turmeric (Curcuma longa L. syn. Curcuma domestica Val.) on estrogen hormone levels Rizka Angrainy,1 Aris Citra Wisuda,2 Asita Elengoe,1 Rathimalar Ayakannu,1 Berliana Irianti,1 Manisha,1 Aida Fitria,1 Siti Aisyah,3 Nuriah Arma,3 Heddy4 11School of Nursing and Applied Sciences, Lincoln University College, Petaling Jaya, Selangor Darul Ehsan, Malaysia; 2Nursing Study Program, Sekolah Tinggi Ilmu Kesehatan Bina Husada Palembang, Indonesia; 3Institut Kesehatan Helvetia, Medan, Indonesia; 4Institut Kesehatan Bina Husada, Serang, Indonesia Abstract Estrogen is essential for maintaining the structure and function of the female reproductive system, and its decline during menopause triggers various symptoms. While Hormone Replacement Therapy (HRT) is commonly used, long-term use increases the risk of certain cancers. Consequently, natural alter- natives like turmeric (Curcuma longa L. syn. Curcuma domestica Val.) are being investigated for their phytoestrogenic potential. This study aimed to investigate the effect of 96% ethanol extract of turmeric on estrogen hormone levels in female rats. An experi- mental study was conducted using 15 female Sprague-Dawley rats over 30 days, divided into five groups: control (0 mg/kg BW), contraceptive pill (10 mg/kg BW), and turmeric extract at doses of 25, 50, and 100 mg/kg BW. The extract was administered orally. On day 31, blood serum was collected, and estrogen levels were measured using the Enzyme-Linked Immunosorbent Assay (ELISA). Estrogen levels increased in all treatment groups com- pared to the control (647 ng/L), with levels of 691 ng/L (25 mg/kg), 709 ng/L (50 mg/kg), and 617 ng/L (100 mg/kg). However, these differences were not statistically significant. Turmeric extract showed a dose-dependent effect, with moderate doses indicating phytoestrogenic benefits. The decline at high doses suggests a dual action-beneficial at moderate levels, inhibitory at excess. Despite non-significant results, the trends support turmeric’s potential as a natural HRT alternative, warrant- ing further studies with larger cohorts and mechanistic evaluations to define optimal dosage, safety, and reproductive implications. Introduction Estrogen refers to a group of steroid hormones that are funda- mental to the development, regulation, and maintenance of the female reproductive system and secondary sexual characteristics.1 Although primarily associated with female physiology, estrogen is also present in males, albeit in significantly lower concentrations.2 Hormones in general are endogenous biochemical messengers synthesized by glands to control and coordinate functions across various tissues and organs.3 They are critical in regulating growth, metabolism, reproduction, and mood.4 A comprehensive under- standing of hormonal roles, especially that of estrogen, is essential for women to navigate the complex physiological transitions across their lifespan.5 Estrogen exists in three primary forms estra- diol, estriol, and estrone each dominant during distinct life stages.6 Estradiol predominates during the reproductive years, estriol dur- ing pregnancy, and estrone during the premenopausal transition.7 Physiologically, estrogen performs a multitude of vital func- tions. It assists in thermoregulation, enhances memory, and mod- ulates neural circuits associated with sexual and reproductive behavior. It also plays a role in lipid metabolism, thereby reducing cardiovascular risk by controlling cholesterol levels. 8 Estrogen facilitates ovarian maturation and menstrual cycle initiation, sup- ports uterine development for implantation, promotes breast development and lactation readiness, and helps maintain skeletal Correspondence: Rizka Angrainy, School of Nursing and Applied Sciences, Lincoln University College, Petaling Jaya, Selangor Darul Ehsan, Malaysia. E-mail: rizkaangrainy.ikeshelvetia@gmail.com Key words: Curcuma longa, estrogen hormone, ethanol extraction, phy- toestrogen, turmeric extract. Contributions: RA conceptualization, data curation, formal analysis, methodology, validation, visualization, writing – original draft, review and editing; AE, RA conceptualization, investigation, methodology, val- idation, and writing – original draft, review and editing; ACW, BI con- ceptualization, methodology, formal analysis, validation, and writing – original draft, review & editing; MA, AF methodology, visualization, resources, investigation, and writing – review and editing. Conflict of interest: the authors declare no conflict of interest. Ethics approval and consent to participate: the research has obtained eth- ical approval from the Medical and Health Research Ethics Commission, Faculty of Medicine, Andalas University, based on ethical certificate No. 22/UN.16.2/KEP-FK/2024. Throughout the research process, the researcher adhered to the principles of information ethics, including con- sent, respect for human rights, beneficence, and non-maleficence. Patient consent for publication: written informed consent was obtained for anonymized patient information to be published in this article. Funding: this research did not receive external funding. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Acknowledgments: I would like to thank my Supervisor for valuable insights and contributions to this study. Received: 21 May 2025. Accepted: 28 August 2025. Early access: 17 October 2025. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2025 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2025; 13:14021 doi:10.4081/hls.2025.14021 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organi- zations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. [page 270] [Healthcare in Low-resource Settings 2025;13:14021] integrity.9 Puberty-related estrogen surges contribute to breast growth and the development of pubic and axillary hair10 During pregnancy, elevated estrogen levels support angiogenesis and nutrient transport to the fetus.11-13 Additionally, increased estrogen may cause vaginal secretions in early gestation. As women approach menopause, hormonal fluctuations culminate in a steady decline, resulting in various physiological and psychological symptoms.14 To alleviate menopausal symptoms resulting from estrogen deficiency, Hormone Replacement Therapy (HRT) is frequently employed. A study by Amanda J. Welton et al. demonstrated that daily oral administration of conjugated equine estrogen (0.625 mg) with medroxyprogesterone acetate (2.5/5.0 mg) over a one-year period significantly reduced vasomotor symptoms, joint pain, sleep disturbances, and vaginal dryness, thereby enhancing post- menopausal quality of life, although some patients reported side effects such as breast tenderness and discharge.15 However, research by Judith K. Ockene, PhD, MEd, indicated that cessation of this combination therapy led to the recurrence of symptoms including vasomotor instability and joint stiffness.16 Despite its therapeutic benefits, long-term HRT use is associated with increased risks of breast, endometrial, and ovarian cancers.17 This risk profile underscores the importance of exploring safer alterna- tives, including phytotherapy, with turmeric (Curcuma longa Linn. syn. Curcuma domestica Val.) being a prominent candidate.18 Turmeric is a perennial rhizomatous herb from the Zingiberaceae family, indigenous to South Asia and now cultivated globally in subtropical climates.19 The dried rhizome is processed into turmeric powder, which has been traditionally utilized across Asia for culinary and medicinal purposes.20 Among its active con- stituents is turmerone a sesquiterpenoid compound also known as α-turmerone that exhibits anti-inflammatory, antioxidant, and neu- roprotective activities.21 These properties may contribute to reduc- ing systemic inflammation, mitigating oxidative cellular damage, and enhancing cognitive resilience.22 Turmerone, particularly con- centrated in ethanol-based turmeric extracts, is of growing interest in therapeutic research. A growing body of evidence supports turmeric’s broad phar- macological potential. Kusuma Dewi’s study found a significant reduction in body temperature in DPT-vaccinated rats treated with turmeric extract.23 Similar antipyretic effects were observed in New Zealand white rabbits.24 Antibacterial assays also reveal turmeric’s greater efficacy against Gram-positive bacteria (e.g., Bacillus sp., Staphylococcus aureus) compared to Gram-negative strains (e.g., Shigella dysenteriae, E. coli), likely due to differential cell wall structures. 25 26 27 Ethanol is recognized as an efficient sol- vent in turmeric extraction due to its high polarity, allowing for optimal isolation of active compounds.28-30 In reproductive health, turmeric has shown the ability to inhibit ovulation and hormonal secretion (FSH, LH, estrogen, progesterone), disrupt the estrous cycle, and induce biochemical changes in uterine fluid – demon- strating reversible antifertility effects in both males and females.31,32 Materials and Methods Tools and materials In alignment with the experimental framework of this study, which involved quantifying estrogen levels in rat serum following turmeric extract administration, the tools and materials were selected to ensure precise biochemical measurement and con- trolled laboratory conditions. The tools utilized included a Bio-Rad xMark™ Microplate Absorbance Reader integrated with Microplate Manager software for accurate absorbance detection during ELISA analysis, a 27.3°C incubator to maintain stable envi- ronmental conditions during sample incubation, absorbent paper, precision micropipettes for accurate liquid handling, and sterile disposable pipette tips to prevent cross-contamination. The prima- ry materials consisted of components from the Bioassay Technology Laboratory ELISA Kit specifically designed for estro- gen quantification, including a standard estrogen solution, an ELISA plate pre-coated with specific antibodies, a standard dilu- ent, Streptavidin-HRP conjugate, substrate solutions A and B, stop solution, wash buffer, and adhesive plate sealers. Supporting bio- logical materials included blood serum obtained from female Sprague-Dawley rats and turmeric ethanol extract (Curcuma longa Linn. syn. Curcuma domestica Val.) prepared using 96% ethanol, with all tools and materials selected to ensure methodological con- sistency and accuracy in detecting potential phytoestrogenic effects of turmeric. Preparation of turmeric extract Turmeric rhizomes were obtained from the Riau region and processed into powder form. The powder was refined through a 60-mesh sieve to achieve a uniform particle size, ensuring extract consistency. Extraction was performed using the maceration tech- nique, whereby the powder was soaked in 96% ethanol to allow the diffusion of bioactive compounds. The mixture was then filtered, and the filtrate was concentrated using appropriate methods before being stored under controlled conditions until use in the treatment phase. Phytochemical screening To determine the chemical profile of the turmeric extract, qual- itative phytochemical screening was conducted to identify sec- ondary metabolites with potential pharmacological activity. Alkaloids were detected using Dragendorff’s reagent, producing an orange precipitate in positive samples. Flavonoids were identi- fied via the Shinoda test, where the addition of magnesium powder and hydrochloric acid resulted in a red or pink coloration. Tannins were tested using ferric chloride (FeCl3) solution, which produced a blue-black or greenish color in the presence of tannins. Saponins were assessed through a froth test, in which persistent foam forma- tion indicated a positive result. Triterpenoids were identified using the Liebermann–Burchard reaction, showing a reddish-brown color change, while steroids were confirmed through acetic anhy- dride–sulfuric acid testing, yielding a blue or green coloration. These procedures provided a qualitative assessment of the key bioactive compounds in turmeric, many of which are associated with antioxidant, anti-inflammatory, and hormonal regulatory activities. ELISA preparation Before beginning the ELISA procedure, all reagents were allowed to reach room temperature to ensure optimal reaction per- formance. A standard curve was generated by serially diluting the estrogen standard solution into six concentrations: 2400, 1200, 600, 300, 150, and 75 ng/L. For each well, 50 µL of either standard solution or serum sample was pipetted, followed by the addition of assay-specific reagents as per the manufacturer’s protocol. All standards and samples were analyzed in duplicate to enhance mea- surement reliability and reduce experimental variability. The wash buffer, provided in concentrated form, was reconstituted according Article [Healthcare in Low-resource Settings 2025;13:14021] [page 271] to the manufacturer’s instructions. Each assay plate underwent five wash cycles, with 0.35 mL of diluted wash buffer per well, to min- imize background interference and improve assay precision. Experimental design and procedure This study was conducted based on the principles of true experimental design, which emphasizes random allocation, con- trolled interventions, and the use of comparison groups to establish causal relationships.14 The dose–response concept guided the selection of turmeric extract concentrations, allowing evaluation of its potential phytoestrogenic effects across a range of doses.15 Fifteen healthy female Sprague-Dawley rats were randomly assigned to five treatment groups (n=3 per group). Group 1 received no treatment (negative control), Group 2 was adminis- tered 1.8 mg/kg Body Weight (BW) of a standard contraceptive pill as a positive control, and Groups 3, 4, and 5 were given turmeric ethanol extract at doses of 25 mg/kg BW, 50 mg/kg BW, and 100 mg/kg BW, respectively. All treatments were delivered orally in a volume of 1 mL daily for 30 consecutive days. On the 31st day, surgical procedures were performed to collect blood samples, and the serum was separated for estrogen hormone analysis. ELISA procedure and observation Estrogen levels in serum were quantified using ELISA, with all standards and samples run in duplicates to ensure reliability. Each well received 50µL of either standard or rat serum, followed by 50 µL of Streptavidin-HRP. After a 60-minute incubation at 27.3 °C, wells were washed five times with 0.35 mL of wash buffer. Subsequently, 50 µL each of Substrate A and Substrate B were added, incubated for 10 minutes in the dark, and the reaction was stopped with 50 µL of stop solution. Absorbance was mea- sured at 450 nm, and estrogen concentrations were determined from the standard curve. Results Phytochemical screening of turmeric ethanol extract Phytochemical screening was conducted to identify the pres- ence of various secondary metabolites in the ethanol extract of turmeric (Curcuma longa Linn. syn. Curcuma domestica Val.). The qualitative analysis focused on six major compound groups, name- ly alkaloids, flavonoids, tannins, saponins, triterpenoids, and steroids. The results are summarized in Table 1. From Table 1, it is evident that the turmeric ethanol extract contains alkaloids, flavonoids, tannins, saponins, and triterpenoids, while steroids were not detected. Standard curve of estrogen concentration vs opti- cal density To quantify estrogen hormone levels, a standard curve was generated using seven known concentrations of estrogen standard solutions. The Optical Density (OD) readings corresponding to each concentration were recorded to develop a calibration curve, as shown in Table 2. Based on Table 2, the standard curve exhibited a strong linear relationship with a correlation coefficient (r) of 0.998. This calibration was used to calculate estrogen concentra- tions in rat serum samples based on their OD readings. Estrogen hormone levels in rat serum after treat- ment The ELISA test was conducted to determine the estrogen hor- mone levels in the serum of female Sprague-Dawley rats after 30 days of treatment. Rats were divided into five groups: negative control (K-), positive control (K+), and three treatment groups receiving turmeric ethanol extract at doses of 25 mg/kg BW (P1), 50 mg/kg BW (P2), and 100 mg/kg BW (P3). An additional com- parison group included female rats mated with males and given turmeric (BK). The results of the ELISA test are presented in Table 3 and visualized in Figure 1. From Table 3 and Figure 1, it can be observed that estrogen levels increased in rats treated with turmer- ic ethanol extract at 25 mg/kg BW (P1) and 50 mg/kg BW (P2), reaching 691 ng/L and 709 ng/L, respectively both higher than the control groups. However, the group receiving the highest dose (100 mg/kg BW, P3) exhibited a lower estrogen level (617 ng/L), suggesting a possible biphasic dose response. The positive control group (K+) and the BK group both showed similar estrogen levels (669 ng/L), while the untreated control group (K-) had the lowest baseline value at 647 ng/L. Discussion The results of this study indicate that the ethanol extract of turmeric (Curcuma longa Linn. syn. Curcuma domestica Val.) con- tains various secondary metabolites, including alkaloids, flavonoids, tannins, saponins, and triterpenoids, but not steroids. These active compounds may contribute to the observed increase in estrogen hormone levels in the tested female rats, as suggested by previous literature reporting that flavonoids can exhibit estro- genic activity by binding to estrogen receptors,36 while certain alkaloids have been shown to influence endocrine function.38 This is supported by the ELISA test results, which showed that treat- ment groups receiving turmeric ethanol extract at doses of 25 mg/kg BW and 50 mg/kg BW had higher average estrogen levels Article Table 1. Phytochemical screening results of turmeric ethanol extract. No Compound Test criteria Test result Interpretation group 1 Alkaloid Formation of white precipitate with Mayer’s reagent White precipitate observed Positive 2 Flavonoid Color change to orange, pink, or red Orange color turned red Positive 3 Tannin Color change to blackish-green or dark blue Orange color turned blackish-green Positive 4 Saponins Persistent foam within 5 minutes Stable foam formation observed Positive 5 Triterpenoid Formation of a brownish or violet ring at phase interface Brownish ring observed at the border of the solution Positive 6 Steroid Formation of greenish-blue ring No greenish-blue ring observed Negative [page 272] [Healthcare in Low-resource Settings 2025;13:14021] (691 ng/L and 709 ng/L, respectively) compared to the negative control group (647 ng/L). However, at the highest dose (100 mg/kg BW), estrogen levels decreased to 617 ng/L, suggesting a possible toxic or inhibitory effect at this concentration. According to Suprihatin, flavonoids and alkaloids in plants can act as abortifacients, potentially causing miscarriage or distur- bances in reproductive processes.33 This is further supported by findings from Ningsih,34 stating that flavonoid and alkaloid com- pounds can disrupt cell membranes, alter membrane components, and inhibit cell division by impairing the formation of membranes responsible for nutrient transport and cellular energy metabolism. In reproductive terms, the permeability of egg and embryo cell membranes is crucial for embryonic growth and development (cleavage).35 The impact of alkaloids on the membranes of egg and embryo cells results in membrane shrinkage, leading to decreased mem- brane integrity, which impairs the development of the egg and embryo cells, potentially resulting in embryonic death. Moreover, saponins, which are characterized by their foaming properties, also negatively impact animal reproduction. They are known to act as abortifacients, inhibit zygote formation, and prevent implantation. Saponins exhibit cytotoxic effects, particularly on developing cells such as those undergoing oogenesis.36 This study also aligns with Ockene, who states that distur- bances during pregnancy can be caused by both internal and exter- nal factors. Internal factors include chromosomal abnormalities, while external factors may involve exposure to viruses, radiation, malnutrition, and chemical substances such as alkaloids, steroids, and alcohol.16 Therefore, the active compounds in turmeric may exert reproductive effects through both hormonal and cellular pathways. Interestingly, although the treatment groups receiving turmeric extract showed increased estrogen levels compared to the negative control, these levels remained lower than those in the pos- itive control group receiving contraceptive pills (average 669 ng/L). This suggests that while turmeric extract may act as a natu- ral phytoestrogen, its potency is still lower than that of synthetic estrogen found in oral contraceptives. Overall, these data suggest that turmeric ethanol extract has the potential to enhance estrogen hormone levels in animal models, particularly at moderate doses (25-50 mg/kgBW). The decline in estrogen levels at the highest dose (100 mg/kgBW) may indicate toxicity or a physiological compensatory response to excessive exposure to active com- pounds. Therefore, based on these findings, the researchers recom- mend further studies using higher dose variations and long-term observations to determine the optimal effect and potential toxicity of turmeric ethanol extract.33 Conclusions The administration of 96% ethanol extract of turmeric (Curcuma longa Linn. syn. Curcuma domestica Val.) was found to effectively increase estrogen hormone levels, with the highest ele- vation observed at a dose of 50 mg/kg body weight, surpassing even the levels found in the positive control group that received birth control pills. This suggests that turmeric extract at moderate doses may have phytoestrogenic activity capable of enhancing endogenous estrogen production. However, at a higher dose of 100 mg/kg body weight, a significant decline in estrogen levels was observed, falling below both the positive control and the untreated negative control group. This indicates that excessively high doses may exert an inhibitory or potentially toxic effect on hormone reg- ulation. Therefore, it can be concluded that while turmeric extract Article [Healthcare in Low-resource Settings 2025;13:14021] [page 273] Table 2. Standard estrogen concentration vs optical density. No. Estrogen Level (ng/L) OD (Optical Density) 1 2400 0.075 2 1200 0.181 3 600 0.503 4 300 0.855 5 150 1.106 6 75 1.240 7 0 1.395 Table 3. Estrogen hormone levels in female rats after treatment. Group Description Mean Estrogen Level (ng/L) BK Female rats mated with males and given turmeric 669 K+ Positive control (contraceptive pill 1.8 mg/kg BW) 669 K- Negative control (no treatment) 647 P1 Turmeric extract 25 mg/kg BW 691 P2 Turmeric extract 50 mg/kg BW 709 P3 Turmeric extract 100 mg/kg BW 617 Figure 1. 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