Steroidal Saponins from Water Eggplant (Fruits of Solanum torvum) Exhibit Anti-Epileptic Activity against Pentylenetetrazole-Induced Seizure Model in Zebrafish

The fruits of Solanum torvum Swartz, a wild relative of eggplant, are consumed as a wild vegetable in tropical regions of Africa, Asia, and South America. In traditional Chinese medicine, it is believed to have anti-inflammatory and sedative effects. In the Philippines, water decoction is used to treat hyperactivity disorder. Twenty-two steroidal saponins were isolated and purified from the fruits grown in Yunnan, China, including six new compounds: torvosides U–Z (1–6). During drying and cooking, the saponins may undergo transformation, resulting in small amounts of sapogenins. These transformations can include dehydration of hydroxyl groups at position C22, formation of double bonds at position 20, 22 or 22, 23, and even formation of peroxide products. Saponin compounds torvoside X (4), torvoside Y (5), torvoside A (7), and (25S)-3-oxo-5α-spirostan-6α-yl-O-β-d-xylopyranoside (20), which are glycosylated at C-6, showed certain anti-epileptic activity in a pentylenetetrazole-induced zebrafish seizure model. No antiproliferative activity was detected when tested on the cancer cell line HepG2, and no hepatotoxic effect was noted on normal liver cell line LO2.


Introduction
Solanum torvum Swartz is a subshrub from the Solanaceae family, which is widespread in tropical regions of Africa, Asia, and South America.The fruit of S. torvum, commonly known as water eggplant (also turkey berry, bhankatiya, sundakkai, kudanekayi), is an edible vegetable variety, which is rich in various beneficial components.In Yunnan Province, located in southwestern China, as well as in southeast Asian countries, such as Thailand, Malaysia, and the Philippines, many ethnic minority areas have a long-standing tradition of consuming young fruits of S. torvum.For instance, the Va people in Yunnan use bamboo pestles to mash the fruit into a paste, which is then seasoned with salt and chili to make a side dish.Similarly, the Jingpo people in Ruili, Yunnan, cook the fruit with pickled bamboo shoots and seasoning, or crush the cooked fruit and mix it with pickled bamboo shoots.In Thailand, fried crispy bhankatiya (the Thai name for the fruit of S. torvum) curry is a popular dish among international tourists.
However, water eggplant is not only used as food.It has long been employed in traditional medicine in Africa and Asia for the prevention and treatment of various ailments [1].The investigation into the chemical constitution of S. torvum can be traced back to the 1940s, with studies revealing the presence of steroidal saponins, flavonoids, organic acids, and other structural classifications [2][3][4][5][6][7].Pharmacological investigations have unveiled the antioxidant [8], antidiabetic [9], antidepressant [10], antihypertensive [11], and antitumor [12] properties of select sterols and sterol glycosides derived from S. torvum.Due to its Molecules 2024, 29, 1316 2 of 15 calming, digestive, hemostatic, and diuretic attributes, S. torvum is frequently employed in traditional medicinal practices in southern China and southeast Asia.
Epilepsy is a chronic neurological disorder, which affects approximately 65 million people worldwide.It is caused by abnormal discharges of groups of neurons, mainly due to an imbalance between excitatory and inhibitory neural transmission [13].Traditional Chinese medicine has been found to have significant efficacy in treating epilepsy, with lower toxicity and fewer side effects, providing a significant advantage over conventional treatments [14].Recent investigations have indicated that the methanol extract of water eggplant, employed as an anti-epileptic and antispasmodic medication in traditional Philippine medicine, contains steroidal saponins, which exhibit anticonvulsant effects [7].Consequently, the research on water eggplant has garnered heightened significance due to the utilization of its medicinal and edible resources.This study aims to isolate and purify the medicinal components of Yunnan water eggplant.The goal is to identify new structures of steroidal saponins, which can effectively treat epilepsy.This could potentially allow for the dual use of water eggplant as both a food and a medicinal product.

Structural Elucidation
The extraction of Solanum torvum fruit was separated using silica gel, octadecylsilyl silica gel (ODS), and preparative HPLC to afford six new steroidal saponins, namely torvosides U-Z (1-6), together with sixteen known compounds (shown in Figure 1).
to its calming, digestive, hemostatic, and diuretic attributes, S. torvum is frequently employed in traditional medicinal practices in southern China and southeast Asia.
Epilepsy is a chronic neurological disorder, which affects approximately 65 million people worldwide.It is caused by abnormal discharges of groups of neurons, mainly due to an imbalance between excitatory and inhibitory neural transmission [13].Traditional Chinese medicine has been found to have significant efficacy in treating epilepsy, with lower toxicity and fewer side effects, providing a significant advantage over conventional treatments [14].Recent investigations have indicated that the methanol extract of water eggplant, employed as an anti-epileptic and antispasmodic medication in traditional Philippine medicine, contains steroidal saponins, which exhibit anticonvulsant effects [7].Consequently, the research on water eggplant has garnered heightened significance due to the utilization of its medicinal and edible resources.This study aims to isolate and purify the medicinal components of Yunnan water eggplant.The goal is to identify new structures of steroidal saponins, which can effectively treat epilepsy.This could potentially allow for the dual use of water eggplant as both a food and a medicinal product.

Screening for Anti-Liver-Cancer Activity
The experimental results demonstrated that the compounds showed no liver toxicity toward HepG 2 cells and normal L02 cells, except for compounds 16 and 17, as determined at a concentration of 70 µM using the MTT assay.This suggests that the aqueous extract of water eggplant contains several steroidal saponins with minimal liver toxicity, making it suitable for consumption (Figure 5).

Discussion
Steroidal saponins, found extensively in plants, have over ten thousand known variants.Their pharmacological activity has garnered global interest.These saponins exhibit significant variance in structure and properties, depending on their source.Furthermore, steroidal saponins from different species of the same plant can display distinct pharmacological activities [14].
In this study, a total of 22 compounds were isolated and purified from the fruit of Solanum torvum using a series of sophisticated separation techniques.These compounds primarily consist of steroidal glycosides with sugar substitutions at the C6 position.Compounds 1-6 are novel furostanol glycosides, characterized by dehydration of the hydroxy

Screening for Anti-Liver-Cancer Activity
The experimental results demonstrated that the compounds showed no liver toxicity toward HepG 2 cells and normal L02 cells, except for compounds 16 and 17, as determined at a concentration of 70 µM using the MTT assay.This suggests that the aqueous extract of water eggplant contains several steroidal saponins with minimal liver toxicity, making it suitable for consumption (Figure 5).that increasing the concentration up to 600 µM significantly reduced the locomotor activity of zebrafish larvae, but further increasing the concentration did not result in additional suppression of movement.Compounds 1-5, 7-8, 10, 15, 20-22 were utilized in the experiment.The results demonstrated that the steroidal saponins torvoside X (4), torvoside Y (5), torvoside A (7), and (25S)-3-oxo-5α-spirostan-6α-yl-O-β-D-xylopyranoside (20) exhibited anti-epileptic activity (Figure 4).Active compounds with a keto group at position C-3 and furan steroid saponins (4, 5, 7, 20) showed better activity.

Screening for Anti-Liver-Cancer Activity
The experimental results demonstrated that the compounds showed no liver toxicity toward HepG 2 cells and normal L02 cells, except for compounds 16 and 17, as determined at a concentration of 70 µM using the MTT assay.This suggests that the aqueous extract of water eggplant contains several steroidal saponins with minimal liver toxicity, making it suitable for consumption (Figure 5).

Discussion
Steroidal saponins, found extensively in plants, have over ten thousand known variants.Their pharmacological activity has garnered global interest.These saponins exhibit significant variance in structure and properties, depending on their source.Furthermore, steroidal saponins from different species of the same plant can display distinct pharmacological activities [14].
In this study, a total of 22 compounds were isolated and purified from the fruit of Solanum torvum using a series of sophisticated separation techniques.These compounds primarily consist of steroidal glycosides with sugar substitutions at the C6 position.Compounds 1-6 are novel furostanol glycosides, characterized by dehydration of the hydroxy

Discussion
Steroidal saponins, found extensively in plants, have over ten thousand known variants.Their pharmacological activity has garnered global interest.These saponins exhibit significant variance in structure and properties, depending on their source.Furthermore, steroidal saponins from different species of the same plant can display distinct pharmacological activities [14].
In this study, a total of 22 compounds were isolated and purified from the fruit of Solanum torvum using a series of sophisticated separation techniques.These compounds primarily consist of steroidal glycosides with sugar substitutions at the C6 position.Compounds 1-6 are novel furostanol glycosides, characterized by dehydration of the hydroxy group at C22, resulting in formation of a double bond at either position 20,22 or 22,23, and in some cases, the formation of peroxide derivatives.Based on the structural characteristics of these compounds, it can be inferred that they are generated as natural products undergo boiling.The C22 position, which exhibits a hemiacetal structure, is inherently unstable and prone to oxidation under normal heating conditions, giving rise to this unique class of compounds isolated in this study.Furthermore, during the isolation process, the hydroxy group at the C22 position of the compounds may undergo methoxylation when exposed to methanol.To prevent such occurrences, a 50% acetonitrile-water solution is commonly employed to revert the hydroxy group back to its original state.
A screening of compounds from water eggplant for anti-epileptic activity revealed that steroidal glycosides (4, 5, 7, 20) with C-6-linked quinovose and rhamnose, a hydroxy group at C-22, a peroxide hydroxy group, or double bonds at C22 and C23 exhibited remarkable anti-epileptic properties.Through the evaluation of hepatotoxicity, spirostanol glycosides (16, 17) with a hydroxy group at position 23 on the F ring were found to possess anti-livercancer activity, and most compounds showed no hepatotoxicity.Some researchers have delved deeper into the structure-activity relationship of steroidal saponins.They found that rhamnose [22] is the crucial sugar unit for pharmacological effects, and the 6-OH [23] compound is also a significant therapeutic site.According to the findings of this study and a review of the literature, most compounds isolated from Solanum torvum contain rhamnose.The 6-OH is replaced by the sugar unit, resulting in different effects.Therefore, this study suggests new possibilities for the active site of steroidal saponins based on these findings.

Plant Material
Fresh fruits of Solanum torvum were harvested between September and October 2017 in the Dehong Dai and Jingpo Autonomous Prefecture of Yunnan Province, China.This variety is known locally as "little bitter fruit" and was identified by Professor Dr. Hai-zhou Li. from the School of Life Science and Technology of Kunming University of Science and Technology.

Extraction and Isolation
Three kilograms of dried water eggplant was pulverized and extracted four times with 70% methanol/water at ambient temperature, resulting in four hundred thirty-five grams of concentrated extract.The extract was subsequently subjected to sequential extraction three times using petroleum ether and dichloromethane.The resulting fractions were concentrated under reduced pressure, yielding the petroleum ether phase (27 g), dichloromethane phase (94 g), and aqueous phase (250 g).A total of 22 compounds were isolated from the aqueous phase utilizing silica gel column chromatography, ODS reverse phase column chromatography, and semi-preparative techniques.Among these compounds, compounds 1-6 were newly identified.

Bioactivity Assays 4.5.1. Anti-Epileptic Activity Studies
The experiment was conducted using 7-day-old zebrafish larvae.Each well of a 48-well plate contained one fish, and each group was replicated 7 times.The zebrafish larvae were observed for their locomotor behavior in a dark environment using a zebrafish behavior tracking analyzer.After a stable period of 10 min, the distance traveled by the larvae within 1 h, with a speed exceeding 20 mm/s, was recorded.The experiment involved testing different concentrations of DMSO, pentylenetetrazole (PTZ), and the positive drug phenytoin sodium.The blank group was treated with 400 µL of E3 water.The effects of different concentrations of DMSO (0.1%, 1%, 3%) were compared, and it was observed that a concentration of 0.1% DMSO had no impact on zebrafish locomotor behavior.In the model group, 380 µL of E3 water was added and pre-incubated for 1 h at 28 • C, followed by the addition of 20 µL of PTZ solution at different concentrations (1.25 mM, 2.5 mM, 5 mM, 10 mM, 20 mM).The results demonstrated that the addition of 20 µL of 10 mM PTZ (final concentration 500 µM) significantly induced rapid locomotion in zebrafish.Therefore, it was determined that a final concentration of 500 µM PTZ is the optimal induction concentration for the zebrafish PTZ-induced seizure model.In the dosing experimental group, after pre-treatment with different concentrations of phenytoin sodium solution (200 µM, 300 µM, 400 µM, 500 µM, 600 µM, 800 µM) for 1 h at 28 • C, followed by the addition of 20 µL of 10 mM PTZ solution, it was observed that phenytoin sodium significantly reduced zebrafish seizure behavior when the concentration exceeded 600 µM.
The primary compounds 1-5, 7-8, 10, 15, 20-22 isolated from the fruit of S. torvum were examined for their anti-epileptic activity.The experiment comprised distinct groups: a blank group, a solvent control group, a model group, a positive drug group, and a compound group.In the blank group, 400 µL of E3 water was administered, while the solvent control group received 400 µL of 0.1% DMSO.The model group was given 380 µL of E3 water; the positive drug group received 380 µL of 600 µM phenytoin sodium solution; and the compound group received 380 µL of 70 µM monomer compound.After a preincubation period at 28 • C for 1 h, 20 µL of 10 mM PTZ solution was added.The movement distance of larvae traveling at a speed exceeding 20 mm/s within 1 h was measured in a dark environment.

Screening for Hepatotoxicity
Cytotoxic activity was evaluated using the MTT assay.HepG2 cells were detached using trypsin during the logarithmic growth phase and seeded into a 96-well plate at a density of 10,000 cells/mL.Each well received 100 µL of cell suspension (containing 1000 cells per well) and was then incubated at 37 • C in a 5% CO 2 incubator for 24 h.After pre-culture, the original culture medium was removed and replaced with 200 µL of the medium containing the respective drugs in each well.In addition, solvent control and positive control groups were also established, with 5 replicate wells per group.The 96-well plate was then incubated at 37 • C in a 5% CO 2 incubator for 72 h.Subsequently, 20 µL of MTT solution was added to each well and incubated at 37 • C for 4 h.Absorbance at 490 nm was measured using a microplate reader (n = 5).The optical density value for each well represents cell viability/proliferation.The same steps were followed for the L02 cell experiment.

Statistical Analysis
The results were expressed as mean ± standard error of the mean (SEM) and subjected to analysis of variance (ANOVA) with post hoc Tukey and Dunnett tests-with 5% significance level-using the GraphPad Prism 5 Demo https://www.graphpad-prism.cn/,accessed on 3 March 2024

Animal Ethics
This study was approved by the animal ethics committee (Kunming University of Science and Technology Experimental Animal Ethics Committee KMUST2023SK04001, approval data is 1 April 2023).