36 Banko Janakari, Vol 35 No. 1 Evaluation of cytotoxicity and antidiabetic activities of plant extracts used in Triphala from Western Nepal using different solvents A. Chataut 1,2, J. Maharjan 1, R. C. Poudel 1, R. Malla 2*, and D. Khadka 1* Plant products have played a vital role in traditional medicine for centuries due to the abundance of secondary metabolites like alkaloids, steroids, flavonoids, terpenoids and tannins. These bioactive compounds are beneficial in addressing various health issues (Chhetri et al., 2008). About 80% of the population in developing countries rely on traditional medicine, including medicinal plants, for primary healthcare needs (WHO, 2013). Although an estimated 250,000 to 500,000 plant species exist globally, only a small fraction has been scientifically investigated for their phytochemical composition and therapeutic potential (Prabhu et al., 2010). Nepal is rich in biodiversity, sheltering over 2,332 species of medicinal and aromatic plants that are extensively used in traditional healing practices (Baral & Kurmi, 2006). These plants are equally significant for both traditional medicine and modern pharmacological applications. The safety of herbal products is often taken for granted, as many people believe that their long history of use without apparent side effects implies that they are inherently safe; however, this belief can be misleading. Just because something is natural or has a long history of use does not guarantee the 1 Molecular Biotechnology Laboratory, Faculty of Science. Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal 2 Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal *Email: rajanimalla200@gmail.com; deegendrakhadka@gmail.com; deegendra.khadka@nast.org.np The Triphala plants: Phyllantus emblica, Terminalia chebula and Terminalia bellirica have been traditionally used in the treatment of various aliments since prehistoric times. Their antidiabetic activity, particularly against α-amylase enzyme, has been reported in studies from different countries. However, limited research has evaluated the antidiabetic potential of Triphala plants originating from Nepal using solvents like hexane, ethylacetate and water. In this study, extraction of the plants was carried out using the Soxhlet method with the solvents. The antidiabetic activity was evaluated through an α-amylase enzyme inhibition assay, while cytotoxic effect was determined through the brine shrimp lethality assay. Among the extracts, the highest percentage yield was obtained from the aqueous extract of T. chebula (7.17%), while the lowest was from the hexane extract of P. emblica (1.28%). The aqueous extracts of T. chebula demonstrated the highest antidiabetic potential with an IC50 value of 97.86 ± 0.17 µg/mL, forming the smallest polygon in the radar diagram, whereas the least potential was exhibited by the hexane extract of P. emblica (IC50 = 810.85 ± 2.05 µg/mL; 0.81 mg/mL), forming the largest polygon in the radar diagram. Regarding safety, the cytotoxicity of these extracts was assessed using the brine shrimp lethality assay. The hexane extract of P. emblica exhibited the least toxicity (LC50 = 8.54 mg/ mL). In contrast, the aqueous extract of T. cheuba showed the highest toxicity with an LC50 of 0.99 mg/mL. Key words: Medicinal plants; Antidiabetic; Brine shrimp lethality; Triphala; Various solvents. Received: 22, December 2024 Revised: 17, March 2025 Accepted: 19, May 2025 Published: 30, May 2025 Banko Janakari, Vol 35 No. 1, 2025 Pp 36-44 https://doi.org/10.3126/banko.v35i1.73002 https://orcid.org/0009-0005-6577-5565 https://orcid.org/0000-0003-3671-9853 https://orcid.org/0000-0002-6603-0822 https://orcid.org/0009-0008-6500-9387 https://orcid.org/0000-0002-2838-1277 37 Banko Janakari, Vol 35 No. 1 absence of harmful effects. Adulteration, improper formulation, and insufficient knowledge about plant- drug interaction can lead to severe adverse reactions. Numerous studies have linked herbal medicines to hepatotoxicity, while other effects such as harm to the kidneys, nervous system, blood, heart, and skin as well as risks of mutation and cancer are also documented (Saad & Said, 2011). Herbal toxicity primarily arises from poor quality control during production, confusion arising from similar plant names and misidentification of plant species. The level of active compounds can vary depending on several factors; the parts of the plant used, the time of harvest, the growth stage and the region and climate where the plant is grown. Herb may also be contaminated with microorganism, fungal toxin (aflatoxins), pesticides, heavy metals, or synthetic drugs (Saad et al., 2006). The identification of the therapeutic and toxic compounds in natural products is cumbersome because of the contamination caused by different harvesting seasons and various extraction protocols used in herbal medication preparation (Opuni et al., 2023). Therefore, scientific validation and safety assessments are essential to ensure the efficacy and safety of herbal products for public health. Diabetes mellitus (DM) is a condition in which blood glucose level exceeds its limit range. It develops when the pancreatic β-cells fail to produce sufficient insulin, when body cells become resilient to insulin or due to a combination of both factors (Khadka & Pandey, 2022). Diabetes is a major risk factor for several complications, including cardiovascular diseases, neuropathy, and retinopathy. According to the International Diabetes Federation (IDF, 2021), an estimated 537 million adults worldwide were living with diabetes in 2021, and this figure is projected to rise to 643 million by 2030 if current trend continues. IDF claims that a significant portion of this increase is occurring in developing countries. In Nepal, diabetes is emerging as a growing public health problem, particularly in urban areas. This is largely attributed to lifestyle and environmental changes such as sedentary activities and dietary shifts. As of 2021, approximately 6.3% of Nepal’s adult population was affected by diabetes (IDF, 2021). The rising prevalence of diabetes places increasing pressure on healthcare systems, necessitating the urgent need for prevention and early intervention strategies. Despite the availability of several synthetic antidiabetic drugs in the global markets, they are not free from severe side effects such as hypoglycemia (sulphonylureas), lactic acidosis and folate and B12 malabsorption (metformin), gastrointestinal symptom (acarbose), weight gain (sulphonylureas and thiazolidinediones), and edema (thiazolidinediones) (Campbell, 2007). Developing antidiabetic drugs without any side effects is still a challenge. Therefore, efforts to discover more secure and effective hypoglycemic agents are continually increasing. Regarding the discovery of natural hypoglycemic agents, our research team focused on testing constituent plants of Triphala, collected from the western regions of Nepal. Triphala is a well-known polyherbal formulation in Ayurveda, composed of three medicinal fruits: P. emblica, T. chebula and T. bellirica. Triphala mixture is prepared by blending equal proportions of the powdered fruits (Venkateswarlu et al., 2019). Owing to its antioxidant, anti-inflammatory, and digestive benefits, Triphala has been widely studied in herbal medicine (Peterson et al., 2017). P. emblica, associated with the family Phyllanthaceae, is indigenous to the Indian subcontinent and its primary chemical constituents include vitamin C, tannins, flavonoid, and phenolic compounds (Prananda et al., 2023). P. emblica has been used traditionally for treating digestive disorders, diabetes, inflammation and for enhancing immunity (Prananda et al., 2023). T. bellirica, a member of the family Combretaceae, is commonly found in Nepal and India. It contains tannins, gallic acid and ellagic acid as its major components. Traditionally, T. bellirica has been used for treating respiratory issues, supporting digestive health and as a mild laxative (Gupta et al., 2020b). Likewise, T. chebula, also from the family Combretaceae, is native to south Asia, particularly, India, Nepal and Sri Lanka. The major chemical components are chebulinic acid, ellagic acid and gallic acid (Muhammad et al., 2012). T. chebula has long been used in traditional medicine for treating digestive issues, respiratory ailments, and promoting would healing (Bag et al., 2013). In this study, the fruit pulp of each plant was subjected to extraction using three different solvents; water, ethylacetate and hexane to identify the most effective extract based on its half-maximal inhibitory concentration (IC50) against α-amylase for diabetes management. Detailed information about the plant samples is presented in Table 1. The choice of solvent plays a crucial role in determining the type and quantity of bioactive compounds extracted (Harborne, 1998). Non-polar solvents like hexane Chataut et al. 38 Banko Janakari, Vol 35 No. 1 extract fat-soluble compounds while polar solvents like water isolate polar compounds and moderately polar solvent such as ethyl acetate extracts a wide range of bioactive compounds including polyphenols, flavonoids, alkaloids due to its intermediate polarity (Cowan, 1999). This approach facilitates the exploration of diverse bioactive compounds in Triphala plants of Nepal origin. Materials and methods Collection and authentication of plant material Plant samples were collected in April 2019 from Mahendranagar, Kanchapur, Nepal, situated at an altitude of 210 meters above sea level, to ensure seasonal consistency. The collected samples were identified by Dr. R. C. Poudel, Senior Scientist at the Nepal Academy of Science and Technology (NAST). The collected materials were air-dried until a consistent weight was achieved. All the samples were collected with the necessary permission from local authorities and in compliance with ethical guidelines to ensure sustainable and responsible sampling practices. Preparation of plant extracts The collected fruits were briefly cleaned with 70% ethanol to remove surface impurities and then shade- dried until a stable weight was achieved. The dried materials were ground into a fine powder using an electric grinder. Soxhlet extraction method was employed to isolate the bioactive compounds using three different solvents: hexane, ethyl acetate, and water. For each extraction, approximately 50 grams of powdered material was uniformly packed in a thimble and extracted separately with 350 mL of the respective solvents. The extraction was carried out until the solvent in the siphon tube appeared transparent. The resulting extracts were concentrated using a rotary evaporator to remove the bulk of the solvent and the obtained extract was then transferred to a beaker and heated on a hot plate at 30-40 °C until a semi-solid consistency was achieved. The semi solid extracts were stored at 4 °C for further analysis. The percentage yield of extracts obtained from each solvent is presented in Table 2. Antidiabetic activity The antidiabetic potential of the plant extracts was evaluated using the α-amylase inhibition assay, following a standard protocol with slight modifications (Tamil et al., 2010; Karki et al., 2021). The presence of undigested starch due to enzyme inhibition was identified by the formation of a blue starch-iodine complex measured at 630 nm. 100 µL of a 1% starch solution was pre-incubated at 37 °C for 5 minutes with 50 µL of different concentrations (20, 40, 80, 160 µg/mL) of each plant extract and the standard inhibitor acarbose. Subsequently, 50 µL of a 50 µg/mL α-amylase solution was introduced into each mixture and incubated at 37 °C for 15 minutes. The enzymatic reaction was halted by adding 200 µL of 0.1M hydrochloric acid (HCl), followed by 250 µL of iodine reagent to develop the color. The absorbance of the resulting blue starch- iodine complex was recorded at 630 nm using a UV-Visible spectrophotometer. All experiments were conducted in triplicate and the percentage inhibition of α-amylase activity was calculated using the following formula: % Inhibition = [1-(Abs2-Abs1/Abs4-Abs3)] ×100 where, Abs1 = Absorbance of a mixture incubated with plant extract, starch and amylase. Abs2 = Absorbance of a mixture incubated with Table 1: Detailed information about the studied plants in the study respiratory issues, supporting digestive health and as a mild laxative (Gupta et al., 2020b). Likewise, T. chebula, also from the family Combretaceae, is native to south Asia, particularly, India, Nepal and Sri Lanka. The major chemical components are chebulinic acid, ellagic acid and gallic acid (Muhammad et al., 2012). T. chebula has long been used in traditional medicine for treating digestive issues, respiratory ailments, and promoting would healing (Bag et al., 2013). In this study, the fruit pulp of each plant was subjected to extraction using three different solvents; water, ethylacetate and hexane to identify the most effective extract based on its half-maximal inhibitory concentration (IC��) against -amylase for diabetes management. Detailed information about the plant samples is presented in Table 1. The choice of solvent plays a crucial role in determining the type and quantity of bioactive compounds extracted (Harborne, 1998). Non-polar solvents like hexane extract fat-soluble compounds while polar solvents like water isolate polar compounds and moderately polar solvent such as ethyl acetate extracts a wide range of bioactive compounds including polyphenols, flavonoids, alkaloids due to its intermediate polarity (Cowan, 1999). This approach facilitates the exploration of diverse bioactive compounds in Triphala plants of Nepal origin. Table 1: Detailed information about the studied plants in the study Scientific name Vernacular name Family Collected sites Parts used Traditional usage Phyllanthus emblica Amala Phyllanthaceae Mahendrnagar, Kanchanpur, Nepal Fruit Rejuvenating agent, tonic for longevity, improve digestion, ailment like respiratory issues sore throat and fever (Prananda et al., 2023) Terminalia chebula Harro Combretaceae Mahendrnagar, Kanchanpur, Nepal Fruit Digestive disorder, wound healing, respiratory disorder. Oral health (Chopra et al., 2023) Terminalia bellirica Barro Combretaceae Mahendrnagar, Kanchanpur, Nepal Fruit Wound healing, liver health, digestive disorder (Gupta et al., 2021) Materials and methods Collection and authentication of plant material Plant samples were collected in April 2019 from Mahendranagar, Kanchapur, Nepal, situated at an altitude of 210 meters above sea level, to ensure seasonal consistency. The collected samples were identified by Dr. R. C. Poudel, Senior Scientist at the Nepal Academy of Science and Chataut et al. 39 Banko Janakari, Vol 35 No. 1 plant extracts and starch only. Abs3 = Absorbance of a mixture incubated with starch and amylase only. Abs4 = Absorbance of a mixture incubated with starch alone. The IC50 value denotes the inhibitor concentration needed to reduce enzyme activity by 50%, A graph was mapped with the extract concentration on the x-axis and percentage inhibition on the y-axis to obtain a linear regression equation. IC50 was calculated through the linear regression by fitting straight line equation with variable slope using Microsoft excel 2019. Brine shrimp lethality assay A pinch of brine shrimp eggs was sprinkled into a beaker filled with artificial seawater and illuminated with a 60 W table lamp at 30 °C for 24 hours to hatch the shrimps. The extracts were initially dissolved in a 1% aqueous dimethyl sulfoxide (DMSO) and subsequently diluted with sea water to obtain test concentrations of 1000 ppm, 100 ppm and 10 ppm. An aliquot of 1 mL of each concentration was transferred into a cleaned sterile measuring cylinder and the volume was raised up to 5 mL using seawater. Twenty nauplii were transferred into each measuring cylinder. The test samples were incubated at room temperature for 24 hours, after which the number of survivors was counted using a pipette. The lethal concentration (LC50) is defined as the concentration causing 50% mortality after 24 hours of exposure along with its 95% confidence intervals. LC50 was determined using the probit analysis method (Finney, 1971). According to Meyer’s toxicity index, extracts with LC50 values below 1000 µg/ml (1 mg/mL) are considered toxic, while those with LC50 values above 1000/ µg/mL are considered as non-toxic (Meyer et al., 1982). Results The extraction yield for the three plants: T. chebula, T. bellirica and P. emblica varied depending on the solvents used (hexane, water and ethyl acetate). Among the solvents, water consistently produced the highest yield, except in the case of T. bellirica. The aqueous extract of T. chebula showed the highest percentage yield (7.16%) followed by P. emblica (7.11%). In comparison, the ethyl acetate extracts yielded moderate yields, with P. emblica yielding 3.69%, T. bellirica 3.62% and T. chebula 3.49%. In contrast, hexane extracts yielded the lowest percentages, ranging from 1.28% in P. emblica to 1.41% in T. chebula, as shown in Table 2. The inhibitory effects of hexane, ethylacetate and water extracts of Triphala plants against α-amylase were evaluated. Acarbose was used as the reference standard, with an IC50 value of 86.49 ± 0.31 µg/mL. The results revealed differences in IC50 values among the various extracts. For P. emblica, the water extract exhibited the lowest IC50 value of 235.22 ± 0.64 µg/ mL (0.235 mg/mL), compared to the hexane and ethyl acetate extracts, as shown in Table 2. Similarly, T. bellirica had IC50 values of 651.05 ± 10.75, 627.12 ± 4.49, and 180.69 ± 0.44 µg/mL for the hexane, ethylacetate and water extracts, respectively. In the case of T. chebula, the aqueous extract had the lowest IC50 value of 97.86 ± 0.17 µg/mL followed by the ethyl acetate extract (163.01 ± 1.8 µg/mL), while the hexane extract showed the highest IC50 value of 500.51 ± 4.33 µg/ml, indicating the least potency. The radar diagram in Figure 1 illustrates that T. chebula exhibited the highest antidiabetic potency of all three solvents, followed by T. bellirica and P emblica, as indicated by the area occupied by the respective polygons. Table 2: Yield percentage and IC50 value of the studied plant in different solvents Name of the plants % yield IC50 ± SEM (µg/mL) Hexane Water Ethylacetate Acarbose Hexane Ethylacetate Water Phyllanthus emblica L. 1.28 7.11 3.69 86.49 ± 0.31 810.85 ± 2.058 656.37± 2.92 235.22 ± 0.64 Terminalia bellirica Retz. 1.32 1.43 3.62 651.05 ± 10.75 627.12±4.49 180.69± 0.44 Terminalia chebula (Gaertn.) Roxb. 1.41 7.16 3.49 500.51 ± 4.33 163.0±1.8 97.86 ± 0.17 The inhibitory effects of hexane, ethylacetate and water extracts of Triphala plants against - amylase were evaluated. Acarbose was used as the reference standard, with an IC�� value of 86.49 ± 0.31 g/mL. The results revealed differences in IC50 values among the various extracts. For P. emblica, the water extract exhibited the lowest IC50 value of 235.22 ± 0.64 g/mL (0.235 mg/mL), compared to the hexane and ethyl acetate extracts, as shown in Table 2. Similarly, T. bellirica had IC50 values of 651.05 ± 10.75, 627.12 ± 4.49, and 180.69 ± 0.44 g/mL for the hexane, ethylacetate and water extracts, respectively. In the case of T. chebula, the aqueous extract had the lowest IC50 value of 97.86 ± 0.17 g/mL followed by the ethyl acetate extract (163.01±1.8 g/mL), while the hexane extract showed the highest IC50 value of 500.51 ± 4.33 g/ml, indicating the least potency. The radar diagram in Figure 1 illustrates that T. chebula exhibited the highest antidiabetic potency of all three solvents, followed by T. bellirica and P emblica, as indicated by the area occupied by the respective polygons. Chataut et al. 40 Banko Janakari, Vol 35 No. 1 Figure 1: Radar diagram showing IC50 in μg/mL of the studied plants in different solvents Cytotoxicity of the extracts was assessed using the brine shrimp lethality (BSL) assay. The LC50 values for different solvents extracts revealed noticeable variations (Table 3). For P. emblica, the aqueous extract exhibited the highest cytotoxicity, with an LC50 value of 1.970 mg/mL, while the ethyl acetate and hexane extracts showed LC50 values of 2.560 mg/mL and 8.54 mg/mL, respectively. T. bellirica displayed LC50 of 1.32 mg/mL in its aqueous extract. Notably, T. chebula’s aqueous extract exhibited the highest cytotoxicity among the tested extracts, with an LC50 of 0.99 mg/mL. According to Meyer’s toxicity index, all extracts except the aqueous extract of T. chebula were considered non-toxic (LC50 > 1 mg/mL). Discussion The choice of solvent significantly influenced the yield percentage of Triphala plant extracts. Aqueous extracts demonstrated the highest yields, with T. chebula yielding 7.17% and P. emblica showing a comparable 7.11%. In contrast, the hexane extracts yielded the lowest, with P. emblica producing just 1.28%, shown in Table 2. These findings suggest that non-polar solvents are less effective in isolating water-soluble phytochemicals like tannins and flavonoids (Nawaz et al., 2020). The high yield observed in aqueous extract aligns with the fact that polar solvents enhance the extraction of hydrophilic bioactive compound (Xia et al., 2023). In comparison, hexane primarily isolates non-polar components like lipids, which are generally less abundant in medicinal plants used for antidiabetic purposes (Sutedja et al., 2020) The moderate yields obtained with ethyl acetate indicate that this solvent is effective for extracting compounds of intermediate polarity, such as phenolic acids and some flavonoids (Baehaki et al., 2020). These results emphasize the importance of solvent-specific extraction methods, as solvent polarity directly affects the composition and quantity of bioactive compounds extracted (Lee et al., 2024). Furthermore, the higher yields in aqueous extracts support their potential for cost-effective and scalable antidiabetic formulations considering the environmental and economic advantages of using water as a solvent (Castro-Puyana et al., 2017). The present study focused on Triphala plants to evaluate their antidiabetic activities using hexane, ethyl acetate and water as extraction solvents. Although extensive research has been conducted on these three plants to evaluate their α-amylase inhibitory activity (antidiabetic) and cytotoxicity using brine shrimp lethality assay, studies specifically using these solvents on Nepal-originating Triphala plants remain scarce. The α-amylase inhibitory activity of P. emblica varies considerably across the literature, with IC50 values ranging from 85.92 µg/mL for seed extract (Dinesh et al., 2016) to 397.67 µg/ mL for methanolic fruit extract (Poongunran et al., 2015), and ethanolic leaf extracts showing 61.12% inhibition under specific conditions (Singh & Kaur, 2015). In our study, the lowest IC50 value observed for P. emblica was 235.22 ± 0.64 µg/mL in the aqueous extract, as compared to its hexane and ethyl acetate extracts (Table 2). Name of the plants % yield IC50 ± SEM (µg/mL) Hexane Water Ethylacetate Acarbose Hexane Ethylacetate Water Phyllanthus emblica L. 1.28 7.11 3.69 86.49 ± 0.31 810.85 ± 2.058 656.37± 2.92 235.22 ± 0.64 Terminalia bellirica Retz. 1.32 1.43 3.62 651.05 ± 10.75 627.12±4.49 180.69± 0.44 Terminalia chebula (Gaertn.) Roxb. 1.41 7.16 3.49 500.51 ± 4.33 163.0±1.8 97.86 ± 0.17 The inhibitory effects of hexane, ethylacetate and water extracts of Triphala plants against - amylase were evaluated. Acarbose was used as the reference standard, with an IC�� value of 86.49 ± 0.31 g/mL. The results revealed differences in IC50 values among the various extracts. For P. emblica, the water extract exhibited the lowest IC50 value of 235.22 ± 0.64 g/mL (0.235 mg/mL), compared to the hexane and ethyl acetate extracts, as shown in Table 2. Similarly, T. bellirica had IC50 values of 651.05 ± 10.75, 627.12 ± 4.49, and 180.69 ± 0.44 g/mL for the hexane, ethylacetate and water extracts, respectively. In the case of T. chebula, the aqueous extract had the lowest IC50 value of 97.86 ± 0.17 g/mL followed by the ethyl acetate extract (163.01±1.8 g/mL), while the hexane extract showed the highest IC50 value of 500.51 ± 4.33 g/ml, indicating the least potency. The radar diagram in Figure 1 illustrates that T. chebula exhibited the highest antidiabetic potency of all three solvents, followed by T. bellirica and P emblica, as indicated by the area occupied by the respective polygons. Figure 1: Radar diagram showing IC50 in µg/mL of the studied plants in different solvents Cytotoxicity of the extracts was assessed using the brine shrimp lethality (BSL) assay. The LC50 values for different solvents extracts revealed noticeable variations (Table 3). For P. emblica, the aqueous extract exhibited the highest cytotoxicity, with an LC50 value of 1.970 mg/mL, while the ethyl acetate and hexane extracts showed LC50 values of 2.560 mg/mL and 8.54 mg/mL, respectively. T. bellirica displayed LC50 of 1.32 mg/mL in its aqueous extract. Notably, T. chebula's aqueous extract exhibited the highest cytotoxicity among the tested extracts, with an LC50 of 0.99 mg/mL. According to Meyer's toxicity index, all extracts except the aqueous extract of T. chebula were considered non-toxic (LC50 > 1mg/mL). Table 3: LC50, slope and regression equation of the studied plants in different solvents Plants Solvent used LC50 mg/mL Slope Regression Equation Phyllanthus emblica Hexane 8.54 R² = 0.9932 y = 0.0053x + 4.7222 Ethylacetate 2.56 R² = 0.9745 y = 0.0188x + 1.3889 Aqueous 1.90 R² = 0.8972 y = 0.0218x + 8.6111 Terminalia bellirica Hexane 7.59 R² = 0.3243 y = 0.006x + 4.4444 Ethylacetate 3.20 R² = 0.9382 y = 0.0135x + 6.6667 Aqueous 1.32 R² = 0.8176 y = 0.0165x + 28.889 Terminalia chebula Hexane 3.20 R2= 0.9382 y = 0.0135x + 6.6667 Ethylacetate 1.12 R² = 0.8176 y = 0.033x + 12.778 Aqueous 0.99 R² = 0.9138 y = 0.0353x + 15.278 Discussion The choice of solvent significantly influenced the yield percentage of Triphala plant extracts. Aqueous extracts demonstrated the highest yields, with T. chebula yielding 7.17 % and P. emblica showing a comparable 7.11%. In contrast, the hexane extracts yielded the lowest, with P. emblica producing just 1.28 %, shown in Table 2. These findings suggest that non-polar solvents are less effective in isolating water-soluble phytochemicals like tannins and flavonoids (Nawaz et al., 2020). The high yield observed in aqueous extract aligns with the fact that polar solvents enhance the extraction of hydrophilic bioactive compound (Xia et al., 2023). In comparison, hexane primarily isolates non-polar components like lipids, which are generally less abundant in medicinal plants used for antidiabetic purposes (Sutedja et al., 2020) The moderate yields obtained with ethyl acetate indicate that this solvent is effective for extracting compounds of intermediate polarity, such as phenolic acids and some flavonoids (Baehaki et al., 2020). These results emphasize the importance of solvent-specific extraction methods, as solvent polarity directly affects the composition and quantity of bioactive compounds extracted (Lee et al., 2024). Furthermore, the higher yields in aqueous extracts support their potential for Table 3: LC50, slope and regression equation of the studied plants in different solvents Chataut et al. 41 Banko Janakari, Vol 35 No. 1 Gupta et al. (2020a) reported that the ethyl acetate extracts of T. bellirica exhibited stronger α-amylase inhibitory activity (IC50 = 43.5 μg/mL) than aqueous extract (IC50 =74.8/ μg/mL). However, in our study, the hexane, ethyl acetate, and aqueous extracts of of T. bellirica exhibited IC50 value of 651.05 ± 10.75, 627.12 ± 4.49 and 180.69 ± 0.44 μg/mL respectively. Similarly, Mukherjee et al. (2010) reported that tannins from T. chebula fruits exhibited 52% inhibition of pancreatic amylase at 100 µg/mL. However, our study found the lowest IC50 in the aqueous extract T. chebula fruit pulp (97.86 ± 0.17 μg/mL), followed by the ethyl acetate extract (163.0 ± 1.8 µg/mL), while the hexane extract demonstrated the highest IC50 value of 500.51 ± 4.33 µg/mL indicating the least potency (Table 2). The radar diagram presented in Figure 1 visually illustrates the comparative potency of three plant extracts in different solvents for antidiabetic activity based on the size of the polygon. The small polygon for T. chebula indicates higher potency across all three solvents, followed by T. bellirica and then P. emblica. Previous studies on the cytotoxicity of P. emblica using the brine shrimp lethality assay have demonstrated potent activity, with LC50 values ranging from 10.25 µg/mL for the chloroform fraction of a crude methanolic extract (Rahman et al., 2009) to 1.25 µg/ mL for seed extract-capped nanoparticles (Dinesh et al., 2017). Similarly, Krishnaraju et al. (2005) reported an LC50 value of 58 μg/mL for the ethanol extract. In comparison, our study revealed that the aqueous extract of P. emblica exhibited the highest toxicity among the three solvents tested, with an LC50 value of 1.38 mg/mL (Table 3). This indicates that the three solvent extracts of P. emblica are non-toxic based on Meyer’s toxicity index. Ali et al. (2013) reported high cytotoxicity for the methanolic bark extract of T. bellirica with an LC50 value of 3.21 mg/ mL. In our findings, the aqueous extract of T. bellirica exhibited the highest cytotoxicity among the tested solvents, with an LC50 value of 1.378 mg/mL, which is still categorized as non-toxic. Previous investigations have demonstrated that ethanol and methanol extracts of T. chebula fruit show cytotoxic activity with LC50 value of 107 μg/ mL (Ved et al., 2010) and 97.36 µg/mL (Sarwar et al., 2013) respectively. However, our study found LC50 values of 0.99, 1.12 and 3.2 mg/mL for the aqueous, ethylacetate and hexane extracts respectively. The aqueous extract exhibited significantly higher toxicity than the other two solvents. The LC50 of the extracts in different solvents are shown in Table 3. According to Meyer’s toxicity index, extracts with LC50 below 1000 µg/ml (1 mg/mL) are considered toxic, while those above 1 mg/mL are considered non-toxic (Meyer et al., 1982). Based on this classification, all three extracts of the Triphala plants prepared using hexane, ethylacetate, and water were found to be non-toxic, except for the aqueous extract of T. chebula, which had an LC50 value of 0.99 mg/mL Supplementary details are presented in Table 3. Conclusion This study demonstrates that Triphala plants as evidenced by their inhibitory effects on α-amylase enzyme, possess promising antidiabetic properties. Among the three Triphala constituent plants, T. chebula demonstrated the strongest activity followed by T. bellirica and P. emblica. While most plant extracts were found to be non-toxic, the aqueous extract of T. chebula showed toxicity, indicating the need for further investigation. Overall, the findings of this study highlight the potential of Nepal-originating Triphala plants as sources for developing natural antidiabetic remedies. The maximum percentage yield of T. chebula in aqueous extract was found. Further pharmacological and toxicological studies are recommended to validate these initial results and to ensure their safe application in clinical settings. Acknowledgments This study was conducted with the support of the Nepal Academy of Science and Technology (NAST). The authors gratefully acknowledge NAST for providing the necessary resources and institutional support to carry out this research. Conflicts of Interest The authors declare that there are no conflicts of interest concerning the research, authorship, and/or publication of this article. Author’s contribution statement A. Chataut: Carried out laboratory experiments, data analysis and methodology. R. Malla: Methodology and Supervision. D. Khadka: Conceived the study, interpreted the data, supervised the work and critically reviewed the manuscript. J. Maharjan: Manuscript revision and data analysis. R. C. Poudel: Collection of plant samples, morphological identification and manuscript revision. Chataut et al. 42 Banko Janakari, Vol 35 No. 1 References Ali, M. S., Faruq, K. O., Islam, A., Nurullah, A. M. M., Chowdhury, K. A. A., & Sayeed, M. A. (2013). Thrombolytic and cytotoxic activities of Terminalia bellerica Roxb. Bangladesh Pharmaceutical Journal, 16 (2), 131-135. https:// doi.org/10.3329/bpj.v16i2.22293 Baehaki, A., Lestari, S., Hendri, M., & Ariska, F. (2020). 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