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INTRODUCTION
Herbal remedies and pharmaceuticals have been effective in the treatment of several diseases. Despite excess of literature on their medicinal properties, there are no standard techniques for characterization of plant materials. Standardization of medicinal plants ensures consistency and therapeutic efficacy. Herbal goods are examined for identifying characteristics, effectiveness, and the purity of the extracts present, because it is vital to evaluate their therapeutic efficacy (Sharma et al., 2021). Solanum melongena L. is a widespread plant around the world, sometimes called as eggplant and aubergine, predominantly in Asian nations, the Horn of Africa, and Mediterranean region. Solanum melongena L. is an commercial pinnacle plant of the family Solanaceae which contains seventy five genera and two thousand plus species and are grown mainly for food and medicinal purposes (Solanke & Tawar, 2019). Eggplants are categorized into three varieties based on their fruit form. It contains high fiber contents, minerals, vitamins C, thiamine, niacin, A, B6, D, B12, E, and K. Eggplant is traditionally utilized for managing a diversed conditions, such as arthritis, bronchitis, asthma, diabetes, and high cholesterol levels (Yarmohammadi et al., 2021). Eggplant is a major vegetable crop grown for its fruits only. It is also called as brinjal. It is a non-tuberous species of high agronomic and economic significance. In 2017, global eggplant production reached 52.3 million tons, making it the world’s sixth most-produced vegetable after tomatoes, onions, cucumbers, gherkins, and cabbages, conferring to the UN’s Food and Agriculture Organization (FAO) (Alam & Salimullah, 2021). Due to the large utility and production, we have not found scientific study on the leaf and stem of this plant. Therefore, the present study planned to summarize the pharmacognostic features of leaf and stem and to find out the antioxidant, antidiarrheal and cardiac potential of eggplant along with networking and molecular docking.
MATERIALS AND METHODS
Chemicals and equipment’s
Dragandroff’s reagent, mayer’s reagent, hager’s reagent, wagner’s reagent, Folin-Cicalto reagent, tannic acid, FeCl3 solution, gelatin solution, basic lead acetate, sodium hydroxide solution, 5% (w/v) glacial acetic acid, 5% (w/v) sodium nitrate solution, DPPH, Hydrogen peroxide and phosphomolybdenum were used for identification studies. Calcium chloride (CaCl2), Sodium chloride (NaCl), Glucose (C6H12O6), Potassium chloride (KCl), Magnesium chloride (MgCl2), Sodium bicarbonate (NaHCO3), Magnesium sulfate (MgSO4), Potassium dihydrogen phosphate (KH2PO4), Sodium dihydrogen phosphate (NaH2Po4) was provided by Merck. Sigma Chemical Co. in St. Louis supplied, Atropine sulfate, Losartan potassium, verapamil HCl.
Collection, drying and preparation of extract
The Solanum melongena L. was collected from local fields of district Muzaffargarh, Punjab, Pakistan in the month of April 2023. The plant parts i.e., leaf and stem are cleaned, shade dried and pulverized after complete drying. Then ground material was macerated in ethanol, which was shaken after every 12 hr. After seven days the material was filtered and dried on rotary evaporator at 40ºC. Weight of each dried extract was measured and stored in ambered glass container and stored in biomedical freezer. The percentage yield for leaf 21% and stem 16% was obtained.
Macroscopic Analysis
Macroscopic features such as, size, shape, color, odor, fracture, texture, external markings and other features were studied by using the standard methods described by Evans 2002.
Fluorescence Analysis
Fluorescence analysis of both powders was carried out according to the protocols described by Kitcher et al., 2020.
Proximate Analysis
Association of Analytical Chemists (AOAC) methodologies were used to conduct the proximate composition analysis. Fat content was determined using the Soxhlet fat extraction technique. Moisture content was determined via air oven drying. Crude protein was evaluated using the Micro Kjeldahl technique. Hydrolysis was used to estimate crude fibre, and the ash content was assessed using a muffle furnace (Gaithersburg, 2000).
Phytochemical Evaluation
Phytochemical evaluation of crude extract of Solanum melongena Leaf (SML) and Solanum melongena Stem (SMS) of eggplant was carried out by using standard protocols as described by Agidew, 2022.
Analysis of Total Phenolic Content
It was estimated by technique of McDonalds et al, 2001, with minor changes. Different concentrations of 2.5-40 mL from the stock solution of leaf and stem were obtained in 5 different test tubes. In each tube 2.5 mL of Folin–Ciocalteu Reagent (FCR) and distilled water (final volume up to 10 mL) with 2 mL of sodium carbonate were incorporated. This mixture was left to stand for 30 min. The volume was adjusted to get concentrations that varied between 2.5-4.0 µg/mL with water. The solutions were then permitted to incubate at 30°C (30 min). The absorbance of the solutions was determined at 765 nm in contrast to blank reagent. It was subsequently determined as mg/g Gallic Acid (GAE/g) correspondent employing the subsequent equation (Izuegbuna et al., 2019).
Analysis of total flavonoid contents
Total flavonoid concentration was determined spectrophotometrically via an aluminum chloride colorimetric assay. Different concentrations of 2.5-40 ml after the standard solution were obtained in 5 different test tubes. Additions were made of 0.15 ml of 5% sodium nitrite and 2 mL of purified water. After the solution equilibrated at ambient temperature for 5 min, 0.15 mL of aluminium chloride (10%) was added and the blend was permitted to stand for a further 5 min. Subsequently, 1 mL of 4% NaOH was added, and the total volume was attuned to 5 mL with water. Finally, the samples were incubated for 15 min to allow for full color development. Then the absorbance was assessed at 420 nm. It was expressed as mg/g of quercetin equivalent employing the following equation (Izuegbuna et al., 2019).
Analysis of total tannin contents
It was calculated by Folin–Ciocalteu assay method described by Vijay and Bhambar, 2014.
Different concentrations of 2.5-40 mL from the stock solution of leaf and stem were obtained in 5 different test tubes. To each concentration, 0.5 mL of Folin–Ciocalteu reagent and 1 mL of Na₂CO₃ (4%) were added sequentially. The mixture was subsequently diluted to 10 mL with water. Following an incubation period, absorbance was made at 725 nm. A gallic acid standard curve was made under identical conditions. Results were stated as mg of Gallic Acid Equivalents per gram of analyte (mg/g GAE), calculated using the following equation (Izuegbuna et al., 2019):
Antioxidant Analysis
Total Antioxidant Capacity (TAC)
The antioxidant capacity of the leaf and stem extracts under examination was assessed using a phosphomolybdenum-based assay, which includes the formation of green phosphomolybdate complexes in acidic media. Test samples with a greater absorbance at 645 nm have greater capacity to reduce Mo (VI) to Mo (V), implying a higher antioxidant capability. This is an essential technique since it analyzes the total antioxidant capability of test samples. The results are represented as ascorbic acid equivalents (Bahal et al., 2022).
DPPH radical scavenging assay
The DPPH radical-scavenging capacity of the leaf and stem extracts was measured using the method described by Gupta et al., (2021) with slight modifications. A 1 mL aliquot of 0.135 mM DPPH in methanol was added to 1 mL of either plant extract or gallic acid standard (0.25–40.0 mg/mL). After vortexing, the reaction mixtures were incubated in the dark at room temperature for 30 min. The absorbance was subsequently measured at 517 nm. Vitamin C and gallic acid served as positive controls. The DPPH radical scavenging activity was determined with the following formula:
DPPH % RSA= Abs con - Abs sam/ Abs con ×100
٭where Abs con was the absorbance of DPPH radical + CH3OH; Abs sam was the absorbance of DPPH radical + plant extract or standards.
Hydrogen per-oxide radical scavenging assay
Using the technique outlined by Agrawal M., 2021, Leaf and stem extracts were assessed for hydrogen peroxide scavenging activity using a standard assay (Agrawal et al., 2021). Test solutions were prepared by mixing 4 mL of aqueous plant extract, vitamin C and gallic acid (0.25–40 mg/mL) with 0.6 mL of a 4 mM H₂O₂ solution in phosphate buffer (0.1 M, pH 7.4). Following a 10-min incubation at room temperature, the absorbance was calculated at 230 nm. The ratio of hydrogen peroxide scavenged was determined using the formula below:
٭where Abs con was the absorbance of H2O2 radical + CH3OH; Abs sam was the absorbance of the H2O2 radical + plant extract or standard.
Total Reducing Power (TRP)
The method as reported by Dahdouh, A., 2022 (Dahdouh et al., 2022). A 100 µL aliquot of sample (SML or SMS) was joint with 200 µL of phosphate buffer (pH 6.6) and 250 µL of potassium ferricyanide (1%) in a microcentrifuge tube. The mixture was vortexed and nurtured at 50°C for 20 min. Following incubation, 200 µL of Trichloroacetic Acid (TCA) (10%) was added and centrifugated at 3000 rpm (10 min). 50 µL of 0.1% ferric chloride was taken in respective 96 well plates and then 150 µL of supernatant from above centrifuged mixture and mixed thoroughly. The interpretations were made at 630 nm wavelength on microplate reader. 100 µL of stock solution of ascorbic acid (1 mg/mL) was given as positive control and respective solvent without sample was used as negative control.
Ferric Ion Reducing Antioxidant Potential (FRAP)
An improved method of Pastor, F. T., 2020 (Pastor et al., 2020) was adopted for the FRAP assay. The FRAP working reagent was made fresh by partying acetate buffer, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl, and 20 mM FeCl₃·6H₂O in a 10:1:1 (v/v/v) ratio [25:2.5:2.5 mL] and pre-warmed to 37°C. Stock solutions of acetate buffer, TPTZ, and FeCl₃ were prepared as described previously [or: Acetate buffer (300 mM, pH 3.6) was prepared by dissolving 3.1 g of CH3COONa in water, adding 16 mL of CH3COOH, and regulating the final volume to 1 L. Separate stock solutions of 20 mM FeCl₃ and 10 mM TPTZ in 40 mM HCl were also prepared.] Aqueous extracts of leaves and stems (100 µL) at concentrations ranging from 0.25 to 40.00 mg/mL were allowed to react with 2.9 mL of the pre-warmed FRAP reagent in the dark for 30 min at 37ºC. The absorbance of the resulting ferrous-tripyridyltriazine complex was then calculated at 593 nm. A normal curve was created using ferrous sulfate (FeSO₄·7H₂O) solutions ranging from 200 to 1000 µM. The reducing antioxidant capacity was expressed as micromoles of ferrous equivalent per gram of dry extract (µM Fe (II)/g extract).
Nutritional Analysis
The A.O.A.C. technique was used to determine the mineral content (Horwitz & Latimer, 2000). 2-4 g of crushed materials have been placed in a crucible and combusted in a muffle furnace (550ºC) for 6 hr. The subsequent ash was liquified in 10 mL of 10% (v/v) Nitric acid (HNO₃) with gentle heating for 20 min. After filtration, the obtained solution was analyzed to determine the mineral content. Ca, Mg, Cu, Se, Cr, Pb, and Zn were measured using an atomic absorption spectrophotometer, whereas Na and K were measured using a flame photometer in the filtrate.
Isolated tissue experimentation
Preparation of animals and housing condition
Albino rabbits (mixed-sex, 1.5–2.0 kg) were sourced from the Animal House of the Faculty of Pharmacy, BZU, Multan. The animals were held under typical laboratory conditions: ambient temperature of 23 ± 3ºC, absolute humidity (30-70%), and a 12-hr light/dark cycle. They were provided with a conventional diet and water ad libitum.
Animals were allowed unrestricted access to water, but they weren’t provided any food the night before the study. After receiving a neck blow, rabbits were killed. The entire research followed all acceptable ethical guidelines outlined by “The Institute of Laboratory Animal Resources, Commission on Life Sciences” (NRC) and is approved by Institutional ethical committee of Bahauddin Zakaria University Multan, letter no 32/UREC/2023 (Council, 2011).
In vitro assays
Bio science isometrical force displacement transducers were connected to the Power lab data collecting system (AD Instruments, Bella Vista, NSW, Australia), which presented the results on a computer running Lab Chart software (version 6). In physiological settings, discrete tissue responses were seen. The percentage variation in the tissue response that was observed following the administration of test dosages was used to quantify the effect of the test drug (Iqbal et al., 2023; Saqib et al., 2021).
Isolated jejunum tissue preparation
Prepare the tissues after the abdominal cavity of rabbits was opened under anesthesia, pieces of the jejunum were dissected and chopped into lengths of about 1 cm. The jejunum fragments were then set vertically in an enclosed organ bath (15 mL volume) filled with Krebs-Henseleit buffer (KHS) having NaCl (118.2 mM), NaHCO3 (25.0 mM), CaCl2 (2.5 mM), KCl (4.7 mM), MgSO4 (1.2 mM), and glucose (11.7 mM). Its higher end was wired to an isometric force transducer, and its lower end was coupled to a tissue holder in the organ bath (UF1 Isometric Transducer). It was kept under 1 g of stress. The KHS was continually oxygenated with a carbogen concoction (95% O2 + 5% CO2) and reserved at a temperature of 37ºC. After stabilization, the tissue preparations were placed under 1g of strain for at least 30 min (e.g., until jejunum contractions became rhythmic). Segments with flimsy contractions were taken out of the protocol after stabilization. Different jejunum segment for every test were used (Chda et al., 2023). On an equilibrated jejunal preparation, the potential spasmolytic and spasmogenic response of crude extracts was investigated through the cumulative addition of various extract dosages of leaf and stem. The response at each dose was normalized to the control contraction and plotted on a dose-response curve. The jejunum was precontracted with 80 mM KCl in order to measure the CCB (Calcium Channel Blockers) activity.
Isolated Aorta tissue preparation
Descending thoracic aortic rings (2–3 mm wide) were prepared and suspended in separate tissue organ baths. The baths contained Krebs-Henseleit solution (37°C) of the following composition (in mM): NaCl 118.2, NaHCO₃ 25.0, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, and glucose 11.7. The solution was continuously bubbled with carbogen (95% O₂ / 5% CO₂). Rings were equilibrated for 50 ± 10 min under an optimal resting tension of 2 g prior to experimentation. Plant extracts were progressively added to a baseline state of repose to better understand any potential vasodilator or vasoconstrictor effects (Janbaz et al., 2015). Losartan and yohimbine were employed as prior treatments on isolated aortic rings to investigate the mechanism of contraction.
In silico approaches
Screening of the active ingredients
We obtained active ingredient information from online databases IMPPAT and Dr. Duke’s Phytochemicals and Ethnobotanical Databases, which is a comprehensive database for active ingredients. We also used MolSoft, SwissADME, and Protox-IΙ, which provide ideal information of the ADME&T properties of natural compounds (Ru et al., 2014). The active compounds were screened based on Bioavailability (OB) and Drug-Likeness (DL), key indices for evaluating ADME characteristics using bioinformatics. Increasing OB can maximize efficacy while minimizing adverse effects (Wang et al., 2017).
Identification of disease target genes
To get the disease targets, the GeneCards and OMIM databases were searched using the keywords “diarrhea, inflammatory bowel disease, constipation and cardiac diseases, i.e. IHD”. The final disease target database was created by de-duplicating the targets in an Excel spreadsheet. The STITCH library was used to express the ultimate gene targets.
Identification of mutual disease effector targets
Using Venny 2.1.0, the active ingredient action targets were intersected with ailments targets to identify shared action targets between the two.
Analysis of Hub Gene and Development of PPI network
The connections between various kinds of mutual target proteins were studied via the String protein association database and Cytoscape software. The CytoHubba plug-in was utilized with network analysis techniques to identify important genes. The Degree method has a stronger prognostic consequence on genes and a greater advantage, so it was utilized in this work to screen key genes for the treatment of diarrhea and cardiac illnesses using the CytoHubba plug-in. Hub genes were found, and the rank file was stored (Liu et al., 2023).
GO and KEGG path investigation
The standard targets were evaluated for the KEGG path and GO function enrichment using SRpolt. The top ten Biological Pathway (BP), Cellular Components (CC), Molecular Functions (MF), and cardiac and diarrhea-related signaling pathway were chosen based on their number of enriched genes.
Molecular docking
We selected the top six chemical compounds and the top three hub genes. To identify potential active compounds, a reverse docking approach was employed. The three-dimensional crystal structures of key protein targets, as identified in the network analysis, were retrieved from the Protein Data Bank. To choose the dynamic elements with the greatest bustle ranking, the structures of important chemicals were retrieved from the PubChem database. AutoDock Tools software was used for adjustments like as hydrogenation and charge balance. The receptor and ligand files were changed to the PDBQT format for docking. AutoDock Vina was used for molecular docking. The resulting poses were visualized and scrutinized using PyMOL software (version 2.2.0).
Statistical Analysis
All experimental data were collected in triplicates and results are expressed as Mean ± SD.
GraphPad Prism version 8 was used to prepare the graphs.
RESULTS AND DISCUSSION
Organoleptic evaluation is important parameter for standardization of herbal drugs, because, the majority of herbal products that are delivered to the market are distorted, twisted, rolled, and shrunken. Therefore, such medications are easily tampered or replaced. The use of pharmacognostic protocols will aid in determining the authenticity of pharmaceuticals because these tests provide results that are unique to a specific drug (Alam & Saqib, 2015). Our results of organoleptic evaluation which will help in proper identification of leaf and stem of Solanum melongena L. and are cited in Table 1.
| Features | Leaf | Stem |
|---|---|---|
| Size | 14 cm | 85 cm |
| Shape | Elliptical | Cylindrical |
| External Color | Brown | Greenish brown |
| Internal color | N/A | Light green |
| Odor | Distinct | Distinct |
| Taste | Pungent | Pungent |
| Apex | Emarginate | N/A |
| Texture | Leathery | Rough |
| Margins | Sinuate | N/A |
| Venation | Reticulate | N/A |
| External markings | N/A | Nodules |
| Surface | Hairy/Pubescent | N/A |
| Fracture | Papery | Fibrous/Short |
| Fracture surface | Irregular | Uneven |
| Petiole size | 1.5cm | N/A |
| Leaf base | Asymmetrical | N/A |
With the aid of contemporary methods and the adoption of appropriate criteria and standards, the World Health Organization (WHO) has addressed the need to ensure quality inspections of medicinal plant materials. Fluorescence analysis and proximate analysis are two standardized parameters used in the current study. The fluorescence analysis is a tool for identifying plant components that provides a clear understanding of the chemical nature. It provides a hint as to whether powders are adulterated, making it a useful diagnostic tool for determining adulteration (Senthilkumar et al., 2019). Our results presented in Table 2 showed different colors both in day light and UV light, forming a bench mark for its authentication.
| Reagent | Leaf | Stem | ||||
|---|---|---|---|---|---|---|
| Day light | 254 nm | 366 nm | Day light | 254 nm | 366 nm | |
| Ethanol | Avo | Avo | Avo | Day | Gre | Gre |
| Methanol | Avo | Avo | Pig | Bai | Pag | Spg |
| Distilled water | Leg | Avo | Bea | Suc | Leg | Sag |
| Dil. H2SO4 | How | Sag | Mog | Van | Leg | Bai |
| Conc. H2SO4 | Reo | Med | Reo | Bea | Goc | Hav |
| Propanolol | Avo | Tar | Pig | Sos | Mig | Sim |
| Chloroform | Leg | Beq | Beq | Bai | Leg | Haw |
| Toluene | Pig | Mog | Avo | Reo | Bai | Wid |
| Dil. HCl | Spl | Tar | Ter | Riy | Haw | Bai |
| Conc. HCl | Hop | Beq | Leg | Pig | Avo | Tar |
| FeCl3 | Reo | Beq | Cha | Riy | Hav | Tar |
| Picric acid | Leg | Hav | Reo | Riy | Hop | Ham |
| Dil. NH3 | Riy | Beq | Ant | Pig | Sim | Haw |
| Iodine | Spl | Day | Avo | Ham | Ter | Bea |
| Benzene | Bai | Hab | Haw | Gold | Tar | Spl |
| Conc. HNO3 | How | Avo | Bea | Suc | Tar | Mor |
| Petroleum ether | Bag | Bea | Avo | Riy | Mdg | Ant |
| NaOH | Lim | Spl | Men | Wid | Lim | Ham |
| Acetic acid | Avo | Wid | Row | Coy | Haw | Tar |
| Acetone | Tar | Bea | Men | Hop | Gold | Med |
| Ethyl acetate | Leg | Hop | Tar | Sag | Reo | Mob |
In our study stem powder had higher moisture content compared to leaf powder and comparatively equal protein content as presented in Table 3. The ash content of stem is greater than leaf. Similarly, stem had significantly higher fat content compared to leaf, but fiber content of both parts was not significantly different as presented in Table 3. The large variance in proximate composition amongst the samples investigated showed that they have a comparative advantage over one another. Both leaf and stem have low moisture content. That leads a lengthy shelf life. The plant has a low protein content. Plants with low levels of crude protein make for less nutrient-dense foliage for creatures with high protein requirements. Low-fat components are also present in the plant. Consuming a great deal of crude fat can lead to health problems including cardiovascular diseases, atherosclerosis, cancer, and aging, thus it’s best to consume it in moderation (Antia et al., 2006). Ash content as a percentage serves as an indicator for the inorganic mineral components identified through research. The findings demonstrated that total ash is high than water insoluble ash but lower than acid insoluble ash. The abundance of minerals in the plants is shown by their high ash content. It contains fibers on higher level, which improves peristalsis, and helps in digestion and used to lowers blood cholesterol levels, which decreases the risk of heart attacks and strokes (Iheanacho & Udebuani, 2009).
| Parameters % | Leaf | Stem |
|---|---|---|
| Moisture | 12.9±0.00 | 14.51±0.01 |
| Protein | 5.99±0.00 | 5.55±0.01 |
| Ash | 6.78±0.01 | 7.53±0.01 |
| Water soluble ash | 3.1±0.05 | 4.00±0.04 |
| Acid insoluble ash | 9.81±0.02 | 12.00±0.02 |
| Fat | 9.99±0.02 | 10.55±0.01 |
| Fiber | 34±0.02 | 37±0.01 |
Phytochemical compounds like, alkaloids, tannins, flavonoids, phenols and saponins are very important in the dealing of different diseases such as cancer, antimicrobial, inflammation and anti-helminthic. In our study both, leaf and stem extracts showed the presence of these metabolites which indicates the therapeutic potential of plant (Table 4).
| Class | Tests | Leaf | Stem |
|---|---|---|---|
| Alkaloids | Dragendorff’s test | ++ | + |
| Mayer’s test | ++ | + | |
| Hager’s test | ++ | + | |
| Wagner’s test | ++ | + | |
| Tannic acid test | ++ | + | |
| Tannins | FeCl3 test | + | + |
| Gelatin test | ++ | + | |
| Flavonoids | Lead acetate test | ++ | + |
| Alkaline reagent test | ++ | + | |
| Phenols | FeCl3 test | ++ | + |
| Ellagic acid test | + | + | |
| Saponins | Froth formation | - | - |
Micronutrients are needed by humans and other organisms in varying levels throughout their lifetimes in order to regulate a wide range of physiological processes.
In our study Solanum melongena L. leaf and stem contains a good amount of Ca, and presence of Mg, K, Na, Cu, Zn, as presented in Table 5. The presence of these essential nutrients showed that leaf and stem can serve as a source of these nutrients in nutrient deficient individuals. The growth of bones and teeth depends on calcium. Additionally, it aids in the production of bodily fluids such as blood and extracellular and intracellular fluids (Olayiwola et al., 2009). Magnesium and zinc are a crucial mineral element they serve as a cofactor of numerous enzymes. The presence of sodium and potassium in a reasonable amount is noteworthy because it aids in the generation of osmotic pressure between the cell and the tissues around it in the body and also controls fluid exchange between the cell and those tissues (Long et al., 2007). Low amount of copper can cause high blood pressure and infertility. Selenium is a trace element that both people and animals require to survive (Junior & Dantas, 2016).
| Mineral/Elemental | Leaf | Stem |
|---|---|---|
| Calcium (Ca) % | 5.82 | 3.42 |
| Magnesium (Mg) % | 0.02 | 0.029 |
| Potassium (K) % | 0.372 | 0.362 |
| Sodium (Na) % | 0.2207 | 0.0113 |
| Copper (Cu) (ppm) | 17 | 2 |
| Zinc (Zn) (ppm) | 0.03 | 0.09 |
| Lead (Pb) (ppm) | 0.06 | 0.05 |
Quantitative analysis showed prominent results for the presence of phenolics, tannins and flavonoids as showed in Figure 1. Antioxidants have grown in popularity due to their ability to protect food and pharmaceutical items from oxidative degradation. Screening for antioxidant capabilities of plants and plant-derived elements demands the use of appropriate practices to investigate the mechanism of antioxidant activity and emphasis on the kinetics of events involving antioxidants. The DPPH radical is an inert organic compound, making it a powerful reagent for analyzing the free radical-scavenging powers of various substances (Sánchez-Moreno, 2002). The extracts showed good scavenging activity as showed in Figure 2A. Most enzymes are capable of being completely inactivated by hydrogen peroxide via oxidizing critical thiol (-SH) groups (PhD, 1999). H2O2 scavenging is relatively higher as showed in results Figure 2B. The TAC (phosphomolybdenum approach) relies on the creation of a Green Mo(V) complex with a maximum absorbance at 695 nm after Mo (VI) is reduced to Mo(V) by antioxidant chemicals. The extracts showed appreciable scavenging power in Figure 2C. The existence of reductants, which have been demonstrated to exercise antioxidant action by disrupting the free radical chain by surrendering a hydrogen atom, is necessary for a chemical to have reducing ability. The reduction of the ferric ion (Fe³⁺) in the potassium ferricyanide multiplex to the ferrous state (Fe²⁺) by antioxidants serves as the basis for measurement. Our results showed a correlation with the standard in Figures 2D & 2E. ‘’Antioxidant power’’ is evaluated using a unique approach called the FRAP assay. A colorful ferrous-tripyridyltriazine multiplex is produced when ferric to ferrous ion drop occurs at a low pH. The results showed that extracts at different concentration showed a linear correlation against standard curve in Figures 2F & 2G.
In silico study
Screening for active compounds
We gathered active ingredient information from online databases. Out of 127 active compounds, only 6 demonstrated good ADMET characteristics (Figure 3). The supplemental materials include comprehensive data on active substances.
Screening of disease target genes
We employed the STITCH and Swiss target prediction database to evaluate prospective Solanum melongena L. targets. A total of 432 targets were discovered (refer to Supplementary Materials). Among these targets included Daturaolone (100), Ascorbic acid (46), Nicotinic acid (76), N-trans-p-Coumaroyloctopamine (100), riboflavin (10), and OPC-4:0 (100). Gene Cards and OMIM database were used to assess 41761 targets for diarrhea, IBD, constipation and heart disorders (see Supplementary Material). Top network candidates were constructed using the active chemicals and targets identified above.
Screening for common disease effector targets
The Venn plot was constructed between compounds targets and genes which showed 284 common unique elements, the overall unique elements are 24996, as showed in Figure 4.
Analysis of Hub Gene and Construction of PPI Network
We searched 432 similar molecules and 24993 targets related with diarrhea and heart disease. The union of chemical and disease targets produced 284 targets (Figure 4). The found targets were used as potential therapeutic targets. These 284 targets have been transmitted to the STRING database, and active protein interactions were picked from databases. The results have been uploaded into Cytoscape for further analysis. The top ten hub genes identified using CytoHubba’s degree technique were TNF, EGFR, STAT3, HIF1A, ESR1, JUN, PPARG, HSP90AA1, PTGS2, and MAPK3. The hub gene is a key target in various biological processes (Zhang et al., 2020). Cytoscape software was used to analyze the results, and Figure 5 depicts the resulting network which shows Daturaolone has 1 node and 100 edges, Ascorbic acid has 1 node and 46 edge, Nicotinic acid has 1 node and 76 edges, N-trans-p-Coumaroyloctopamine has 1 node and 100 edges. The top three genes i.e. TNF has 1 node and 121 edges, EGFR has 1 node and 102 edges and STAT3 has 1 node and 91 edges.
GO and KEGG Pathway Analyses
The hub genes were entered into the STRING library for GO and KEGG pathway enrichment analysis. Supplementary material displays the whole GO enrichment analysis results, whereas Figure 7 shows the top ten results for the three aspects of BP, CC and MF. Biological Processes (BP) are primarily focused on (Figures 6 & 7 A) responding to cellular response to chemical stress, hemostasis, peptidyl−serine modification, peptidyl−serine phosphorylation, regulation of blood pressure, regulation of small molecule metabolic process, response to lipopolysaccharide, response to molecule of bacterial origin, rhythmic process and steroid metabolic process. Cell Component (CC) analysis (Figures 6 & 7 B) revealed that the targets mostly focused on azurophil granule lumen, caveola, cytoplasmic vesicle lumen, membrane microdomain, membrane raft, membrane region, nuclear envelope lumen, plasma membrane raft, secretory granule lumen and vesicle lumen. The Molecular Function (MF) study identified targets (Figures 6 & 7 C) that were closely connected to dioxygenase activity, icosanoid receptor activity, iron ion binding, ligand−activated transcription factor activity, nuclear receptor activity, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, prostaglandin receptor activity, prostanoid receptor activity, protein serine/threonine kinase activity and steroid binding.
The KEGG analysis findings are shown in the supplementary material. Figures 6 & 7 D depicts the 10 most prominent signal pathways. KEGG pathway enrichment analysis showed that target genes were implicated in many pathways, such as, Aldosterone−regulated sodium reabsorption, Bladder cancer, EGFR tyrosine kinase inhibitor resistance, HIF−1 signaling pathway, Insulin resistance, the PD-1/PD-L1 checkpoint pathway in cancer, proteoglycans in cancer, renal cell carcinoma, thyroid hormone signaling pathway and VEGF signaling pathway. Thus, Solanum melongena leaf and stem extract was found to regulate several pathways, potentially affecting GIT and CVS.
Molecular docking
Molecular docking investigations indicate that all six active components engage to the targets with a certain stability. Solanum melongena L. active constituents bind more effectively to target proteins, coexist with manifold interactions, and have a substantial contest with their active sites. These components may be key targets for treating diarrhea and cardiac problems. Different interactions with gene TNF (Figure 8), EGFR (Figure 9), STAT3 (Figure 10) are displayed while binding energies and H-bonding are elaborated in Table 6.
| Compound | Genes | Pose | Binding energy | Hydrogen bonds | Hydrophilic bonds |
|---|---|---|---|---|---|
| Daturaolone | TNF | 1 | -8.2 | Arg214 | Arg30, Thr228, Ala251, Ser171, Lys252, Leu229, Phe172, Ile150, Gly274, Asp275, Val227. |
| EGFR | 1 | -8.7 | Glu711 | Arg705, Trp731, Pro733, Leu707, Lys708, Ile706, Glu931, Arg932, Pro934, Leu933, Ile918, Tyr915, Asp916, Glu734, Glu736. | |
| STAT3 | 6 | -5.1 | Lys57 | Pro32, Arg58, Lys31, Ser54, Glu61, Leu18, | |
| OPC_40 (Oxypinone) | TNF | 3 | -5.2 | Lys216, Arg214, Leu229. | Thr228, Val227, Gln148, Lys252, Ile250, Ala251, Ser171, Phe230, Asp275, Ile150. |
| EGFR | 1 | -4 | Gln812, Ser811. | Gln976, Arg973, Pro975, His988, Met987, Asp984, Tyr813, Gly810. | |
| STAT3 | 1 | -3.8 | Lys109, Val102. | Lys21, Tyr25, His22, Lys98, Asp101, Phe105, Arg106. | |
| N-trans-p-Coumaroyloctopamine | TNF | 1 | -6.4 | Lys252, Leu229, Ser171. | Arg214, Phe172, Ile250, Thr228, Asp275, Arg30, Arg231, Asp273, Gly274, Ile150, Phe230, Gln148, Ala251. |
| EGFR | 4 | -5.6 | Tyr827, Ser768, Cys939. | Val769, Arg831, Leu703, Pro937, Pro936, Gln935, Ala702, Arg776, Asp770. | |
| STAT3 | 3 | -5.9 | Asp101, Gln100, Arg76. | Tyr83, Glu104, Phe103, Phe79, Ser80. | |
| Nicotinic acid | TNF | 7 | -3.9 | Arg30, Gln213, Leu226, Thr228. | Leu224, Val227, Asp222, Leu211. |
| EGFR | 3 | -4.4 | Arg836, Tyr869. | Val765, Asp761, Glu762, Gly857, Leu858, Ala859, Lys860. | |
| STAT3 | 7 | -3.1 | Leu290, Gln288, Gln291. | Ser292, Phe289. | |
| Ascorbic acid | TNF | 3 | -4.6 | Thr157, Ile270, Ala268, Gln269, Asn267. | Pro156, Thr155, Leu272, |
| EGFR | 1 | -5 | Asp770, Lys852, Glu1015. | Pro772, Asn771, Arg776, Val774, Cys775, Gln791. | |
| STAT3 | 1 | -3.5 | Glu61, Arg58. | Leu18, Lys57, Ser54, Thr55. | |
| Protein tyrosine phasphatase | TNF | 1 | 6 | Asp275, Ile250, Lys252, Gln148, Ser171. | Gly274, Ile150, Leu229, Ala251, Phe172, Phe230, Val227, Thr228. |
| EGFR | 4 | -5.8 | Lys745, Asp855, Oun1797, Arg841. | Asn842, Phe723, Val726, Leu747, Gly857, Thr854. | |
| STAT3 | 1 | 5.2 | Lys109, Arg106, Asp101, Lys98. | His22, Tyr25, Leu99, Val102, Phe105 |
Isolated tissue experimentation
The extracts of Solanum melongena L. leaf and stem were tested on isolated jejunum tissue samples. The jejunum is utilized because smooth muscles are highly reactive.
Solanum melongena Stem (SMS) produced a spasmolytic reaction when administered to spontaneously constricting isolated jejunum tissue at cumulative doses ranging from 0.003 to 3 mg/mL, with an EC50 value of 0.3722 mg/mL (95% CI: 0.1561 to 0.9179, n=3). To evaluate relaxant activity, tissue preparations were pre-contracted with a high-K+ (80 mM) solution to induce depolarization. Subsequent cumulative addition of the stem extract (SMS) induced a complete, concentration-dependent relaxation of these evoked contractions (0.003–3 mg/mL). The half-maximal Effective Concentration (EC₅₀) was 0.1372 mg/mL (95% confidence interval: 0.05469–0.3296; n=3) (Figure 11A). Solanum melongena Leaf (SML) increased contractile response, but its spasmogenic action was inhibited by atropine (1 μM) pretreatment (Figure 11 B).
Isolated tissue is used for the research of underlying mechanism since it is untouched by nervous or hormonal stimuli and only retorts spontaneously. A brief increase or decrease in free Ca++ in the cytosol is the main trigger for smooth muscle spontaneous contraction. This easily accessible cytosolic Ca++ intermingles with the muscle’s contractile elements to produce a momentary activation or deactivation of those elements. Antispasmodic medications treat conditions with hyperactive gastrointestinal symptoms by disrupting this system (Gilani et al., 2006) Several studies have indicated that the Ca++ antagonistic consequence of curative herbs is the fundamental source of their means of action (Saqib & Janbaz, 2021). The SMS extract reduced spontaneous contractions at doses of 3 mg/mL, via inhibiting calcium input into the cell. To further investigate the anti-spasmodic method of action of stem extract, rabbit jejunum tissue was subjected to protracted constriction with high-K+ (80 mM). Elevated extracellular potassium (K+) concentrations are known to induce sustained smooth muscle contraction. This occurs through the depolarization of the membrane potential, which activates Voltage-Dependent Calcium Channels (VDCCs). The subsequent influx of free Calcium ions (Ca²+) into the cytosol triggers and maintains the contractile response (Iqbal et al., 2023). Stem extract relaxed high k induced contractions at doses 0.003-3 mg/mL (Figure 11A). This result shows that Ca++ channel blockers exist in plants because they are useful in situations where the gut is hyperactive (Saqib & Janbaz, 2021). Leaf extract produced spasmogenic response at doses range from 0.1-1 mg/mL which was masked when tissue was pretreated with Atropine, a muscarinic blocker (Figure 11B). Studies suggest that activation of the M3 muscarinic receptor stimulates PLC, which in turn promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the secondary messenger’s inositol 1,4,5-trisphosphate (IP3) and Diacylglycerol (DAG). The IP₃-induced release of Ca²+ from the SR elevates cytosolic Ca²+. This ion serves a dual role: it cooperates with DAG to activate Protein Kinase C (PKC) and, more directly, binds to calmodulin. The resulting Ca²+/calmodulin complex activates Myosin Light-Chain Kinase (MLCK), which phosphorylates Myosin Light Chains (MLCs) to promote contraction. A contractile response is then produced by an interaction network between phosphorylated MLCs and actin (Wahid et al., 2021).
Furthermore, Solanum melongena L. leaf and stem extract demonstrated contractile action on isolated aortic tissues in tissue bath concentration range of 0.03-5.0 mg/mL and 0.01-5 mg/mL, which was found to be minimized when isolated rabbit aortic preparations were pre-treated with yohimbine for stem extract (Figure 12A) and was clinched to be arbitrated through activation of α-adrenergic receptors and pretreatment with losartan reduced leaf extract activity, which was attributed to activation of angiotensin II receptors (Figure 12B) (Bolton, 1979).
The antidiarrheal activity observed in the isolated jejunum assay suggests that several bioactive compounds previously identified in Solanum melongena L.—such as daturaolone, ascorbic acid, riboflavin, nicotinic acid, luteolin, and oxypinone—may contribute to this effect. The pharmacological basis for this activity likely involves interactions with key regulatory pathways in gut motility. Specifically, modulation of calcium channel function and inhibition of H+/K+-ATPase could underlie the extract’s antimotility effect, potentially by reducing intracellular calcium influx and altering acid secretion, thereby diminishing peristaltic movement (Qazi et al., 2022). The known antidiarrheal mechanisms of these compounds involve the dual inhibition of Cyclooxygenase (COX) and Lipoxygenase (LOX) enzymes, coupled with calcium channel blocking activity, all of which contribute to reduced intestinal motility and secretion. (Wahid et al., 2022). Luteolin has been reported to exert antidiarrheal activity by reducing the volume and weight of small intestinal contents. This effect may be mediated through the upregulation of Na+/K+-ATPase and Creatine Kinase (CK) activity and gene expression, thereby enhancing electrolyte reabsorption and normalizing intestinal ion concentrations (Dong et al., 2021). Recent research indicates that flavonoids and phenolic acids—including quercetin, luteolin, nicotinic acid, and ferulic acid—may have therapeutic potential against diarrheal disorders. Their proposed mechanisms involve the modulation of key pathways in gut smooth muscle contraction, such as inhibition of L-type voltage-gated calcium channels, antagonism of M3 muscarinic receptors, or interference with downstream signaling proteins like Phospholipase C (PLC). These findings collectively suggest that the antidiarrheal activity of Solanum melongena L. is likely mediated through a combination of antisecretory and antimotility actions.
This aligns with the established multi-modal mechanisms of many medicinal plant extracts, which have been shown to alleviate diarrhea through one or more pathways, including anti-secretory effects, enhanced intestinal absorption, reduced gut motility, antimicrobial activity, and direct antispasmodic action. The therapeutic relevance of these mechanisms has been consistently demonstrated in various in vivo studies (Rawat et al., 2017) and are related to our results.
CONCLUSION
Pharmacognostic studies concluded in our research showed that leaf and stem contain different types of chemical compounds and minerals/micronutrients which are responsible for therapeutic potential. Furthermore, the antidiarrheal effects of Solanum melongena L. parts were produced by regulatory genes of calcium-mediated smooth contraction and contractile effect on aorta was mediated by angiotensin ΙΙ receptors.