Annona squamosa and Tetracapidium conophorum exhibited dose ratio dependent additive and synergic combination effects in in-vitro and in-vivo models of hyperthyroidism

 Corresponding author: Mbagwu Ikechukwu Sonne Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Annona squamosa and Tetracapidium conophorum exhibited dose ratio dependent additive and synergic combination effects in in-vitro and in-vivo models of hyperthyroidism Kingsley Obumneme Onwuegbusi 1, Mbagwu Ikechukwu Sonne 1, *, Ajaghaku Amara Anwuchaepe 3, 6, Ajaghaku Daniel Lotanna 2, Chinwuba Paul 5 and Okoro-Akpandu Elizabeth 4 1 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Agulu, Anambra State, Nigeria. 2 Department of Pharmacology, Faculty of Pharmaceutical Sciences, Enugu State University of Science and Technology, Enugu State, Nigeria. 3 Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, David Umahi Federal University of Health Sciences, Uburu, Ebonyi State, Nigeria. 4 Department of Pharmacology and Toxicology, David Umahi Federal University of Health Sciences, Uburu, Ebonyi State, Nigeria. 5 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, P.M.B 410001, Nsukka, Enugu State, Nigeria. 6 International Institute for Pharmaceutical Research and Innovation, David Umahi Federal University of Health Sciences, Uburu, Ebonyi State, Nigeria. World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 Publication history: Received on 24 April 2025; revised on 07 June 2025; accepted on 09 June 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.3.2192 Abstract Annona squamosa and Tetracapidium conophorum leaf extracts have been documented to possess anti-hyperthyroidal activity and are used together in ethnomedicine; however, their effective combination dose ratios have not been explored. This study investigated the combined therapeutic effects of Annona squamosa and Tetracapidium conophorum extracts in in vitro and in vivo models of hyperthyroidism. In vitro studies showed concentration-dependent inhibition of thyroid peroxidase (TPO) enzyme activity by both extracts, with Tetracapidium conophorum exhibiting a lower EC50 (224.05 μg/ml) than that of Annona squamosa (437.24 μg/ml). combination of half their EC50 produced 51% TPO inhibition and a combination index (CI) of 1.0, indicating additivity, while 250 μg/ml Annona squamosa combined with 65 μg/ml Tetracapidium conophorum resulted in 57% inhibition and a CI of 0.8, suggesting synergism. In an in vivo hyperthyroidism model induced by L-thyroxine, both extracts dose-dependently increased thyroid-stimulating hormone (TSH) levels. The combination dose ratios of 30 mg/kg Annona squamosa + 90 mg/kg Tetracapidium conophorum and 25 mg/kg Annona squamosa + 105 mg/kg Tetracapidium conophorum produced a CI of 0.7, indicating synergism. The combination treatment also significantly reduced elevated T3 and T4 levels, liver enzymes, and lipid peroxidation while improving kidney function markers. Annona squamosa and Tetracapidium conophorum extracts exhibited both additive and synergistic effects at different dose ratios in hyperthyroidism models, highlighting their potential to enhance antithyroid efficacy through strategic combinations. Keywords: Hyperthyroidism; Drug interaction; Combination therapy; Annona squamosa; Tetracapidium conophorum; Thyroid peroxidase; Synergism World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1178 1. Introduction Hyperthyroidism, also known as overactive thyroid, is a clinical condition characterized by excessive production of thyroid hormones (triiodothyronine, T3, and tetraiodothyronine, T4) by the thyroid gland, leading to systemic hypermetabolism and increased oxidative stress [1]. Hyperthyroidism affects a notable proportion of the population, with a higher prevalence in women and younger adults [2]. The thyroid gland is one of the most common endocrine disorders in the world (generally about 4% to 7% of thyroid nodules are symptomatic). Approximately half of the cases of thyroid disease involve hyperthyroidism, and the other half involves hypothyroidism [3]. Untreated hyperthyroidism can lead to cardiovascular dysfunction, atrial fibrillation, embolic events, fragility fractures, weight loss, and osteoporosis [4]. Conventional therapeutic options for hyperthyroidism, including antithyroid medication, radioiodine, and surgery, have not improved over the past 70 years, despite substantial unmet clinical needs and a significant lack of efficacy for many patients [5]. Relapse occurs in over half of the adults after discontinuation of antithyroid drugs, and the lack of functional thyroid tissue following definitive treatment with radioiodine or surgery results in hypothyroidism with a lifelong requirement for levothyroxine replacement and associated clinical and biochemical monitoring [6]. Serious side effects, including agranulocytosis, hepatotoxicity, and vasculitis, have also been associated with current antithyroidal medications [7]. Conventional treatment resistance has been described for the management of Graves’ disease-associated hypertyroidism [5]. Such cases are characterized by the persistence of thyrotoxicosis and hyperthyroidism in patients on an optimal dose of antithyroidal drugs for at least 6 months, with good patient compliance and at least one substitution of the antithyroidal drug type [6]. Drug resistance complicates the management of hyperthyroidism, especially in Africa where access to other therapies is often difficult. Further exploration of thyrotoxicosis mechanisms has revealed that the complexity of the disease condition requires multiple approaches [8,9]. It is now clear that the future of antithyroidal therapy relies on effective combination therapies that can tackle diverse hyperthyroidal evolution and resistance pathways. With this new understanding, the next problem is to determine the correct combination [10]. Natural products play an important role in the discovery and development of drugs. Tetracarpidium conophorum and Anona squamosa are among the numerous plants with therapeutic potential against hyperthyrothoxicosis are Tetracarpidium conophorum and Anona squamosal [11]. Annona squamosa L. (Family Annonaceae) is a tropical fruit-bearing tree, also referred to as sugar apple or custard apple. It is highly valued for its many therapeutic applications, including antithyroid, antibacterial, antidiabetic, and anti-inflammatory properties, in addition to its sweet and creamy fruit [12]. Acetogenins found in plants also have anticancer properties, particularly against colon and breast malignancies. It is also used for digestive health and skin conditions, and as a natural sedative. Among these, its use as an antithyroid agent is especially notable [13,14]. According to research on Annona squamosa, this plant has great potential as a natural treatment for hyperthyroidism [11]. Its antihyperthyroid properties are mainly supported by research on its seeds and leaves, which has shown that they can regulate thyroid hormone levels and reduce the negative physiological effects of hyperthyroidism [15,16]. Tertacarpidium conophorum (Family Euphorbiaceae), commonly known as African walnut, is a medicinal plant that is extensively utilized in West African traditional medicine. Plants have been used to treat oxidative stress, thyroid dysfunction, and inflammatory diseases. In hyperthyroidism, excessive thyroid hormone synthesis causes tissue damage and oxidative stress, and its anti-inflammatory and antioxidant properties are especially important. Additionally, studies have demonstrated its capacity to alter endocrine and metabolic processes, which may contribute to thyroid health. The various pharmacological properties of Tetracapidium conophorum, including its historical application in treating thyroid dysfunction symptoms, make it a viable option for additional research on combination treatments for hyperthyroidism [11]. Although individual extracts of Annona squamosa and Tetracarpidium conophorum have demonstrated effectiveness in blocking thyroid peroxidase (TPO), a crucial enzyme in the synthesis of thyroid hormones, the potential of their combinations has been undermined scientifically and may serve as the right key to unlocking successful antithyroidal combination therapy. This study was designed to evaluate the combined therapeutic effects of Anona squamosa and Tetracapidium conophorum leaf extracts in in in-vitro and in-vivo models of hyperthyroidism [11]. 2. Material and methods 2.1. Plant materials Tetracarpidium conophorum and Anona squamosa leaves were obtained during the rainy season (July 2022) from Nsukka Enugu State, Nigeria. The plant materials were authenticated by a taxonomist, Mr. Nwafor Felix, of the Department of Pharmacognosy and Environmental Medicine, University of Nigeria, Nsukka. A voucher specimen was deposited in the herbarium of the Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Agulu. The voucher number for Annona squamosa leaves was PCG/474/A/049, World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1179 and the voucher number for the leaves of Tetracapidium conophorum was PCG/474/E/022. The leaves were air-dried at room temperature and pulverized using a mechanical grinding machine (GX160 Delmar 5.5HP). 2.2. Animals Male Swiss Albino rats of 8 weeks old were used for in vivo pharmacological evaluations, while albino mice (25 – 30 g) were used for the acute toxicity study. To reduce individual variation in disease induction and drug response, the animals were bred in the Animal House of the Department of Pharmacology, Faculty of Pharmaceutical Sciences, Enugu State University of Science and Technology, under ideal conditions of temperature, humidity, and light. The animals were fed pelletized feed (Vital Feeds, Nigeria) and had access to filtered water ad libitum. All animal experiments were conducted in compliance with the NIH Guide for the Care and Use of Laboratory Animals (National Institute of Health (NIH) (2011) Pub No: 85-23). The Ethics Committee at Enugu State University of Science and Technology (ESUT), located in Agbani, Enugu State, approved the study. (Ref No: ESUT/AEC/2024/0356/AP217) 2.3. Extraction Approximately 1kg of pulverized leaves of Tetracarpidium conophorum and Anona squamosa were separately extracted by cold maceration method as outlined by Ajaghaku et al. [17] using 5 litres of 90% methanol. The plant-to-solvent ratio was maintained at 1:5. The mixture was then shaken intermittently for 72 h. The solution was first filtered through a muslin cloth and then through a Whatman filter paper. The filtrate was concentrated to dryness in vacuo using a rotary evaporator (RE300 Model, United Kingdom) at 40 °C. 2.4. Total Phenolic Content of the Extracts by Folin Ciocalteu’s Assay Nzekwe et al. [11] reported the total phenolic contents of Annona quamosa and Tetracapidium conophorum, and the total phenolic content was estimated from the calibrated curve, which was prepared from gallic acid solution and expressed as milligrams of gallic acid equivalent (GAE) per gram of the extracts. 2.5. Phytochemical Analysis Nzekwe et al. [11] reported the methanol extract of Annona quamosa yielded 9.42%, whereas Tetracapidium conophorum yielded 7.56%. While Qualitative phytochemical analysis of extracts from Annona squamosa and Tetracapidium conophorum identified various secondary metabolites, the extract from Annona squamosa contained alkaloids, saponins, cardiac glycosides, tannins, flavonoids, and steroids/terpenoids, but did not contain reducing sugars. In contrast, the extract from Tetracapidium conophorum comprises flavonoids, tannins, and alkaloids but lacks saponins, glycosides, and steroids/terpenoids. 2.6. In Vitro Pharmacological Assays 2.6.1. DPPH assay The free-radical scavenging activity of the plant extracts was evaluated using the method described by Ajaghaku et al. [18]. The inhibitory effects of different dilutions of Annona squamosa (7.8, 15.6, 31.5, 62.5, and 125 ug/ml) and Tetracapidium conophorum (62.5, 125, 250, 500, and 1000 ug/ml) extracts were calculated using the following relationship: DPPH scavenging activity = 100 [(AC – AS)/ AC] • AC = Absorbance of control • AS = Absorbance of sample A plot of the percentage scavenging activity against concentration was used to determine the median effective concentration (EC50s) of the extracts. 2.6.2. Combination Dose selection for DPPH radical scavenging activity Using Loewe Additivity combination strategy equation: (𝑎 𝐴+ 𝑏 𝐵= 1) World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1180 Where A and B are the median effective doses of Annona squamosa and Tetracapidium conophorum extracts, respectively, and a and b represent the doses of these agents (Annona squamosa and Tetracapidium conophorum extracts, respectively) that were combined. complemented with isobologram analysis, all the pairs of doses of drugs A (Annona squamosa extract) and B (Tetracapidium conophorum extract) that could lead to the combined effect of EAB to form additivity were defined, and these were drawn as a line of additivity of negative slope on a graph where the x and y axes represent the doses of drugs A and B. All experimental points below the line that could give the desired effect (50% scavenging of HPPH) corresponded to a CI < 1 and indicated synergism. Combination doses were selected based on the predicted combinations that would provide additivity and synergism. The doses included: a1b1 – a10b10 represented a combination of Annona squamosa and Tetracapidium conophorum extract in the ratios of 8:8, 6.52:84, 6:32, 5:64, 4.42:159.46, 4:40, 3:104, 2:52, 2:148, and 1:200 µg/ml, respectively. The DPPD scavenging effects of these combination doses were then determined. 2.6.3. Thyroid peroxidase inhibitory activity Thyroid Peroxidase enzyme was prepared according to the method described by Habza-Kowalska et al. [19]. Measurements were performed using a plate spectrophotometer (BioTek) in 96 well plates at a wavelength of 470 nm. The assay was conducted as follows: 50 µL of buffer, 40 µL of pure substance solution, methanol extract solution, or in vitro digested solution, 50 µL of guaiacol, 20 µL of thyroid peroxidase enzyme, and 50 µL H2O2. The total volume of the mixture was 210 µl. In the sample probe, the extracts were replaced with a buffer. Absorbance readings were recorded every minute for a total of 3 min at 37 °C, and the unit of TPO activity was defined as the change in the absorbance per minute. All measurements were performed in triplicate. The TPO inhibitory activity will be calculated as follows: %inhibition = (1− (∆A/〖min〗_test)/(∆A〖min〗_blank )) × 100% where ΔA/min is the linear absorbance change per minute of the test material, and ΔA min blank is the linear change in absorbance per minute of blank. The IC50 value was determined by interpolation of the dose-response curves. The IC50 values were calculated using fitted models as the concentration of the tested compound that gave 50% of the maximum inhibition based on a dosedependent mode of action. 2.6.4. Combination Dose selection for Thyroid peroxidase inhibitory activity Loewe Additivity combination strategy equation as previously stated above was used. Combination doses were selected based on the predicted combinations that would provide additivity and synergism. The doses included a1b1 – a10b10 representing a combination of Annona squamosa and Tetracapidium conophorum extract in the ratios of 437:224, 350:15, 275:15, 250:65, 219:112, 175:45, 150:115, 125:75, 100:60, 50:45, and 50:165 µg/ml, respectively. The TPO inhibitory effects of these combination doses were then determined as previously described. 2.7. In vivo Pharmacological Assays 2.7.1. Induction of Hyperthyroidism After acclimatization of the rats for this assay, hyperthyroidism was induced by daily subcutaneous administration of L-thyroxine (T4) (Sigma, USA) at a dose of 600 μg/kg for 12 consecutive days according to the method described by Kim et al. [20]. World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1181 2.7.2. Treatment protocol for determination of effective dose For each extract, the animals were randomly divided into five groups of six rats each after 12 days of L-thyroxine (T4) treatment as follows: Group 1 served as normal control and received 10 ml/kg 5% Tween 80. Group 2 served as the hyperthyroidism control and received distilled water orally for 15 days. Group 3 served as the treatment control and received 10 mg/kg propylthiouracil i. p. (PTU) orally for 15 days. Group 4 served as extract treatment group A and received 100 mg/kg extract orally for 15 days. Group-5 served as extract treatment group B and received 200 mg/kg extract orally for 15 days. Group-6 served as the extract treatment group C and received 400 mg/kg of the extract orally for 15 days. Group 7 served as the extract treatment group D and received 800 mg/kg of the extract orally for 15 days. Group 8 served as extract treatment group E and received 1000 mg/kg of the extract orally for 15 days. The effective doses of the two plants were determined based on their effect on thyroid-stimulating hormone, which formed the basis of dose selection for the combination study. 2.7.3. Pharmacological assays After 15 days of treatment, blood was collected from the retro-orbital plexus puncture of all overnight-fasted rats using a microcapillary. Serum was separated to estimate thyroid-stimulating hormone (TSH) levels. 2.7.4. Serum Thyroid Hormones Serum levels of thyroid-stimulating hormone (TSH) were analyzed by colorimetric competitive enzyme immunoassay using an ELISA kit (Elabscience, USA). The ED50 value was determined by interpolation of the dose-response curves. The ED50 values were calculated using fitted models as the concentration of the tested extracts (Annona squamosa and Tetracapidium conophorum) that gave 50% increase in TSH. 2.7.5. Combination Dose selection for in vivo anti-hyperthyroid activity Loewe Additivity combination strategy equation as previously stated above was used. Combination doses were selected based on the predicted combinations that would provide additivity and synergism. The doses include: a1b1 – a10b10 represented combination of Annona squamosa and Tetracapidium conophorum extract in the ratios of 50:45 mg/kg; 45:30 mg/kg; 35:60 mg/kg; 33.5:153.5 mg/kg; 30:90 mg/kg; 25:105 mg/kg; 15:60 mg/kg; 10:120 mg/kg; 10:210 mg/kg; 5:150 mg/kg respectively. The experimental procedure for the induction and treatment of hyperthyroidism was as previously described. Blood samples were collected into evacuated tubes, and serum was separated by centrifugation at 3000 rpm for 10 min at 4°C. The separated serum was used immediately for analysis. Serum levels of the thyroid hormones T3 and T4 and thyroidstimulating hormone (TSH) were analyzed by colorimetric competitive enzyme immunoassay using an ELISA kit (Elabscience, USA). Further toxicity assays were carried out on groups of animals that received combination doses (33.5:153.5 mg/kg – additivity combination dose; 30:90 mg/kg and 25:105 mg/kg – synergistic combination doses). The effects of the treatment on body weight, liver function, kidney function, and lipid peroxidation were tested. 2.7.6. Biochemical Assay of Serum Liver Marker Enzymes Serum alanine and aspartate aminotransferases were estimated using the method described by [21] using ALT and AST test kits (Span Diagnostics Ltd., India). ALT and AST concentrations were extrapolated from a graph of concentrations against wavelength absorbances of known ALT and AST concentrations. Alkaline phosphatase was estimated by the method described by [21] using an ALP test kit (Span Diagnostics Ltd., India. ALP concentration was calculated using the following equation: 𝐴𝐿𝑃 = Abs of samples x value of standard (IU L) Abs of standard World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1182 Where Standard value = 50 IU/L 2.7.7. Biochemical assay of kidney parameters Serum creatinine and blood urea nitrogen (BUN) levels were estimated using the method described by Tietz [22] and Heinegard and Tiderstrom [23], respectively, using BUN and creatinine test kits (Teco Diagnostics, USA). 2.7.8. Malondialdehyde (MDA) Assay MDA levels in serum were estimated using a modified thiobarbituric acid method as described by Kinsella et al. [24] using a malondialdehyde assay kit (Elabscience Biotechnology Co. Ltd., South Africa). 2.8. Statistical Analysis Numerical data are presented as mean ± standard deviation (SD). The obtained data were analyzed using a one-way ANOVA test followed by Newmann-Keuls multiple range tests. Statistical analyses were performed using the GraphPad version 3.1. P values were considered statistically significant at P < 0.05. 3. Results 3.1. Extraction, yield and phytochemical content Table 1 Yield and Phytochemical content of Annona squamosa and Tetracapidium conophorum Phytocompounds squamosa Tetracapidium conophorum Saponins + - Flavonoids + + Tannins + + Alkaloid + + Glycosides + - Steroid/Terpenoids + - Total phenolic (mgGAE/g) 266.27+14.2 100.05+7.7 Yield (%) 9.42 7.56 Annona squamosa yielded greater extract than Tetracapidium conophorum; however, both extracts were less than 10% of their powdered samples (Table 1). Higher phenolic content was also recorded for Annona squamosa than for Tetracapidium conophorum. Annona squamosa contains an abundance of diverse phytocompounds, whereas saponins, glycosides, and steroids are absent from the phytocompounds identified in Tetracapidium conophorum. 3.2. Combination effect of Annona squamosa and Tetracapidium conophorum on thyroid peroxidase activity Both Annona squamosa and Tetracapidium conophorum (figure 1 and 2) showed concentration-dependent inhibition of the thyroid peroxidase enzyme activity. A higher inhibitory effect was shown by Tetracapidium conophorum compared to Annona squamosa as evidenced by the lower concentration (224.05 ug/ml) of Tetracapidium conophorum required to produce half-maximal inhibitory effect (EC50) compared to almost double the concentration (437.24 ug/ml) required by Annona squamosa to produce the same half-maximal inhibitory effect. Using the EC50s of the extract, all pairs of both extracts that could lead to an additive combination effect were drawn as a line of additivity of negative slope (isobologram) (figure 3). At EC50 of Tetracapidium conophorum, no quantity of Annona squamosa was required to produce half-maximal inhibition of thyroid peroxidase activity and vice versa. However, increasing the concentration of Annona squamosa in combination led to a reduced concentration of Tetracapidium conophorum required to produce half the maximal effect. Figure 4 shows that the combination of both extracts at various dose ratios produces different interactive effects. A combination of half their EC50s (219 ug/ml for Annona squamosa and 112ug/ml for Tetracapidium conophorum) produced 51% inhibitory effect with a combination index of 1.0, an evidence of additive combination interaction. Other combination dose ratios that were tested produced World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1183 subthreshold effect (lower than 50%), except 250 ug/ml of Annona squamosa combined with 65 ug/ml of Tetracapidium conophorum, which produced 57% inhibitory effect and CI of 0.8 – an indication of synergistic interaction. Figure 1 Inhibitory effect of graded concentrations of Tetracapidium conophorum on thyroid peroxidase enzyme activity Figure 2 Inhibitory effect of graded concentrations of Annona squamosa on thyroid peroxidase enzyme activity where a and b represent the combination ratios of Annona squamosa and Tetracapidium conophorum respectively; a1ba = 350:15 ug/ml; a2b2 = 275:15 ug/ml; a3b3 = 250:65 ug/ml; a4b4 219:112 ug/ml; a5b5 = 175:45 ug/ml; a6b6 = 150:115 ug/ml; a7b7 = 125:75 ug/ml; a8b8 = 100:60 ug/ml; a9b9 = 50:45 ug/ml; a10b10 = 50:165 ug/ml. Figure 3 Isobologram plot of combination strategy of Tetracapidium conophorum and Annona squamosa on inhibition of thyroid peroxidase enzyme World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1184 where a and b represent the combination ratios of Annona squamosa and Tetracapidium conophorum respectively; a1ba = 350:15 ug/ml; a2b2 = 275:15 ug/ml; a3b3 = 250:65 ug/ml; a4b4 219:112 ug/ml; a5b5 = 175:45 ug/ml; a6b6 = 150:115 ug/ml; a7b7 = 125:75 ug/ml; a8b8 = 100:60 ug/ml; a9b9 = 50:45 ug/ml; a10b10 = 50:165 ug/ml. Figure 4 Inhibitory effect of combination concentrations of Annona squamosa and Tetracapidium conophorum on thyroid peroxidase enzyme activity 3.3. Combination inhibitory effect of Annona squamosa and Tetracapidium conophorum on DPPH radical Annona squamosa and Tetracapidium conophorum inhibited DPPH free radicals in a concentration-dependent manner. Annona squamosa has better inhibitory potentials with IC50 8.84 ug/ml (fig. 5) compared to Tetracapidium conophorum with IC50 318.92 ug/ml (fig. 6). Using a dose-effect-based strategy of analysis of combination interactions, an isobologram plot was derived using the effective DPPH inhibitory concentrations of both plant extracts, as shown in fig. 7. An additive combination interaction was produced by a 4.42 ug/ml combination of Annona squamosa and 159.46 ug/ml of Tetracapidium conophorum. Similarly, 6.52 ug/ml of Annona squamosa combined with 84 ug/ml of Tetracapidium conophorum produced an additive combination interaction. When traced on the isobologram plot, these combination concentrations fell on the line of additivity, and their combination index was calculated to be 1.0, a confirmatory indication of the additivity combination interaction. The concentrations that produced additivity interactions were able to produce up to 50% inhibition of DPPH radical, which was the target/desired effect. No other combination concentrations tested had an effect up to the threshold or target. Combination concentrations of Annona squamosa less than 4 ug/ml produced inhibition below 40%, as shown in fig. 8. Figure 5 Inhibitory effect of graded concentrations of Annona squamosa on DPPH radical World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1185 Figure 6 Inhibitory effect of graded concentrations of Tetracapidium conophorum on DPPH radical Where a and b represent combination ratios of Annona squamosa and Tetracapidium conophorum respectivelya1ba = 8:8 ug/ml; a2b2 = 6.52:84 ug/ml; a3b3 = 6:32 ug/ml; a4b4 5:64 ug/ml; a5b5 = 4.42:159.46 ug/ml; a6b6 = 4:40 ug/ml; a7b7 = 3:104 ug/ml; a8b8 = 2:52 ug/ml; a9b9 = 2:148 ug/ml; a10b10 = 1:200 ug/ml. Figure 7 Isobologram plot of combination strategy of Tetracapidium conophorum and Annona squamosa on inhibition of DPPH radical Where a and b represent combination ratios of Annona squamosa and Tetracapidium conophorum respectivelya1ba = 8:8 ug/ml; a2b2 = 6.52:84 ug/ml; a3b3 = 6:32 ug/ml; a4b4 5:64 ug/ml; a5b5 = 4.42:159.46 ug/ml; a6b6 = 4:40 ug/ml; a7b7 = 3:104 ug/ml; a8b8 = 2:52 ug/ml; a9b9 = 2:148 ug/ml; a10b10 = 1:200 ug/ml. Figure 8 Inhibitory effect of combination concentrations of Annona squamosa and Tetracapidium conophorum on DPPH radical World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1192 Where a and b represent combination ratios of Annona squamosa and Tetracapidium conophorum respectivelya4b4 33.5:153.5 mg/kg; a5b5 = 30:90 mg/kg; a6b6 = 25:105 mg/kg; PTU = propylthiouracil. * = P<0.05 compared to vehicle control; ♯ = P<0.05 compared to propylthiouracil; ¶ = P<0.05 compared to Naïve control group. Figure 20 Effect of combination doses of Annona squamosa and Tetracapidium conophorum on serum Alkaline phosphatase concentration 3.7. Combination effect of Annona squamosa and Tetracapidium conophorum on kidney function markers in hyperthyroidism-induced animals Where a and b represent combination ratios of Annona squamosa and Tetracapidium conophorum respectivelya4b4 33.5:153.5 mg/kg; a5b5 = 30:90 mg/kg; a6b6 = 25:105 mg/kg; PTU = propylthiouracil. * = P<0.05 compared to vehicle control; ♯ = P<0.05 compared to propylthiouracil; ¶ = P<0.05 compared to Naïve control group Figure 21 Effect of combination doses of Annona squamosa and Tetracapidium conophorum on serum creatinine concentration World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1193 Where a and b represent combination ratios of Annona squamosa and Tetracapidium conophorum respectivelya4b4 33.5:153.5 mg/kg; a5b5 = 30:90 mg/kg; a6b6 = 25:105 mg/kg; PTU = propylthiouracil. * = P<0.05 compared to vehicle control; ♯ = P<0.05 compared to propylthiouracil; ¶ = P<0.05 compared to Naïve control group. Figure 22 Effect of combination doses of Annona squamosa and Tetracapidium conophorum on serum Blood urea nitrogen concentration Figure 21 shows that hyperthyroidism was accompanied by a significant (P<0.05) reduction in serum creatinine concentration and a significant (P<0.05) increase in blood urea nitrogen, as evidenced by the statistical comparison between the naïve uninduced control and vehicle control groups. Although the combination doses did not differ significantly (P>0.05) from each other in terms of their effect on these kidney function biomarkers, only a5b5 produced a significant (P<0.05) increase in serum creatinine concentration compared with the vehicle control group. The effect produced by the reference standard was similar to that of the naïve control group and was significantly higher than that produced by the combination dose ratio of Annona squamosa and Tetracapidium conophorum. For the blood urea nitrogen as shown by fig. 22, treatment with the combination doses produced a significant (P<0.05) reduction in BUN concentration compared to the vehicle control group. The reductive effect of these treatments was able to reduce BUN concentration to a concentration similar to that found in the naïve uninduced control group, similar to the reference drug-treated group. No significant (P>0.05) difference existed between BUN concentration in the combination extract-treated groups and the naïve control group, as well as the reference drug-treated group. 3.8. Combination effect of Annona squamosa and Tetracapidium conophorum lipid peroxidation in hyperthyroidism induced animals As shown in figure 23, hyperthyroidism was marked by an increase in serum malondialdehyde, a product of lipid peroxidation. The increase in serum malondialdehyde in the vehicle control animals was significant (P<0.05) compared to that in the naïve uninduced control. Treatment with the combined doses of Annona squamosa and Tetracapidium conophorum produced a significant (P<0.05) reduction in serum malondialdehyde levels, comparable (similar) to the effect produced by the reference standard PTU 10 (mg/kg). The inhibition of lipid peroxidation by the combined doses did not differ significantly (P>0.05) across the different dose ratios and compared to the reference drug. However, when compared to the naïve uninduced control, both the combination doses and the reference standard showed significantly (P<0.05) higher serum malondialdehyde levels. World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1194 Where a and b represent combination ratios of Annona squamosa and Tetracapidium conophorum respectivelya4b4 33.5:153.5 mg/kg; a5b5 = 30:90 mg/kg; a6b6 = 25:105 mg/kg; PTU = propylthiouracil. * = P<0.05 compared to vehicle control; ♯ = P<0.05 compared to propylthiouracil; ¶ = P<0.05 compared to Naïve control group. Figure 23 Effect of combination doses of Annona squamosa and Tetracapidium conophorum on serum Blood urea nitrogen concentration 3.9. Combination effect of Annona squamosa and Tetracapidium conophorum on body weight in hyperthyroidism induced animals Where a and b represent combination ratios of Annona squamosa and Tetracapidium conophorum respectivelya4b4 33.5:153.5 mg/kg; a5b5 = 30:90 mg/kg; a6b6 = 25:105 mg/kg; PTU = propylthiouracil. * = P<0.05 compared to vehicle control; ♯ = P<0.05 compared to propylthiouracil; ¶ = P<0.05 compared to Naïve control group. Figure 24 Effect of combination doses of Annona squamosa and Tetracapidium conophorum on body weight Induction of hyperthyroidism resulted in body weight reduction, as evidenced by the significant (P<0.05) difference between the pre-induction and pre-treatment body weights across all groups. Although the control group showed a significant (P<0.05) reduction in body weight between the pre-treatment and post-treatment intervals, treatment with the combination doses resulted in a further reduction in body weight, with a non-significant increase in body weight when compared to the pre-treatment values. Compared to the post treatment body weight of the vehicle control group, all the combination dose ratios produced significant (P<0.05) increases in body weight, except for the reference standard treated group. All other treatment groups failed to restore body weight to the pre-induction value, as evidenced by the significant (P<0.05) difference that exists between the pre-induction and post-treatment body weight, as shown in figure 24. World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1195 4. Discussion in recent years, there has been a growing interest in the exploration of natural sources for the development of novel therapeutic agents [25] Several plants have been used in folk medicine throughout the world [26]. Plants have been a source of medicine for the treatment of numerous diseases owing to their diverse phytocompounds. This diversity in plants is better harnessed when used in crude unfractionated forms and combinations [27]. However, it is too simplistic to assume a positive synergy from any two or more herbs combined in traditional practice, as some may lead to an unexpected decrease in activity due to competition for the same site of action or interference with the pharmacokinetics of each other [28]. Similarly, when combined, two plants can exhibit both antagonism and synergism, depending on the dose pair used [29]. The benefit of combination therapy is not simply attributed to the properties of the plants but could also depend on the dose ratio [30]. As the cells do not differentiate between a single dose and a combination, two drugs combined at a given dose ratio could be considered as a third agent with a dose-effect relationship [31]. The establishment of an optimal dose-level pair for each plant extract intended for use in combination therapy is therefore indispensable for maximizing the treatment efficacy of combination therapy. The use of optimal dose pairs in combination therapies increases the chances of improved efficacy and decreases adverse effects [32]. Owing to these advantages, combination therapies have become a standard treatment for several diseases [33]. As such, they continue to represent a promising approach for indications of unmet medical needs. However, combination therapy for the treatment of hyperthyroidism has not been well explored. This motivated the assessment of the combined effects of two antithyroid plants, Annona squamosa and Tetracapidium conophorum, to establish their nature of interaction and effective combination dose ratio. This study investigated the combined therapeutic effects of Annona squamosa and Tetracarpidium conophorum extracts in in vitro and in vivo models of hyperthyroidism. The results demonstrated that these plant extracts exhibit both additive and synergistic effects in inhibiting thyroid peroxidase activity and ameliorating hyperthyroidism, depending on the specific dose ratios used. In vitro studies showed that both Annona squamosa and Tetracapidium conophorum extracts inhibited thyroid peroxidase (TPO) enzyme activity in a concentration-dependent manner. Tetracapidium conophorum exhibited more potent TPO inhibition, with a lower EC50 (224.05 μg/ml) than Annona squamosa (437.24 μg/ml). This suggests that Tetracapidium conophorum may contain compounds with strong direct inhibitory effects on TPO. When combined at certain dose ratios, the extracts produced additive and synergistic inhibition of TPO activity. For example, combining 250 μg/ml Annona squamosa with 65 μg/ml Tetracapidium conophorum resulted in 57% TPO inhibition and a combination index of 0.8, indicating synergism. This highlights the potential for enhanced antithyroid effects through the strategic combination of these plant extracts. The antioxidant activity of the extracts, as measured by DPPH radical scavenging, also showed dose-dependent effects. Interestingly, Annona squamosa demonstrated more potent antioxidant activity than Tetracapidium conophorum despite its weaker TPO inhibition. This suggests that the antithyroid mechanisms of these extracts likely involve multiple pathways beyond direct TPO inhibition, including the modulation of oxidative stress. The combination of extracts at certain ratios produces additive and synergistic antioxidant effects, which may contribute to their overall antithyroid activity. In an in vivo hyperthyroidism model, both extracts showed dose-dependent increases in thyroidstimulating hormone (TSH) levels, with Annona squamosa exhibiting greater potency. This is in contrast with the in vitro TPO inhibition results, in which Tetracapidium conophorum was more potent. This discrepancy may be due to differences in bioavailability, metabolism, or additional mechanisms of action in vivo. Importantly, certain combinations of extracts produced synergistic increases in TSH levels. For instance, combining 30 mg/kg of Annona squamosa with 90 mg/kg of Tetracapidium conophorum resulted in a combination index of 0.7, indicating synergism. This demonstrated the potential for enhanced in vivo antithyroid effects through strategic extract combinations. Combination treatment also effectively reduced elevated T3 and T4 levels in hyperthyroid animals. All tested combinations significantly lowered T3 levels compared to the vehicle control, with some combinations achieving nearly 50% reduction. Similar effects were observed for the T4 levels. These results further support the antithyroid efficacy of the extract combinations. Beyond thyroid hormone modulation, extract combinations showed beneficial effects on other hyperthyroidism-associated parameters. They significantly reduced elevated liver enzymes (ALT, AST, and ALP), improved kidney function markers, and decreased lipid peroxidation as measured by malondialdehyde levels. These findings suggest that combinations of extracts may help mitigate the systemic effects of hyperthyroidism on multiple organ systems. The observed additive and synergistic effects of Annona squamosa and Tetracapidium conophorum extracts likely stem from their diverse phytochemical composition. Both extracts contain phenolic compounds, which are known for their antioxidant properties and potential to modulate thyroid function. The presence of alkaloids, particularly in Annona squamosa, may contribute to additional antithyroid mechanisms. The combination of these diverse compounds from two different plant sources appears to enhance overall antithyroid efficacy through multiple complementary pathways. This study highlights the importance of dose-ratio selection for achieving optimal combination effects. Different ratios of Annona squamosa and Tetracapidium conophorum extracts produced varying degrees of additivity and synergism across the different parameters. This underscores the need for careful optimization of herbal combinations to maximize therapeutic benefits. World Journal of Advanced Research and Reviews, 2025, 26(03), 1177-1198 1196 5. Conclusion This study provides evidence for the potential use of Annona squamosa and Tetracapidium conophorum extract combinations as a novel approach for managing hyperthyroidism. The observed additive and synergistic effects, both in vitro and in vivo, suggest that these plant extracts may offer enhanced antithyroid efficacy compared to their individual use. Furthermore, the multi-target effects on thyroid hormones, oxidative stress, and organ function markers indicate a holistic therapeutic approach. Future research should focus on optimizing extract combinations, elucidating specific bioactive compounds and mechanisms, and evaluating long-term safety and efficacy in clinical settings. Compliance with ethical standards Disclosure of conflict of interest The authors declare that they have no competing interests. Data availability The materials used in this study are described in the references section. 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