Introduction

Natural healing methods have long been integral to global therapeutic practices1, 2. Polyherbal extracts are widely used in South India to treat inflammatory conditions, oxidative stress, and gastrointestinal issues3, 4. One such polyherbal decoction is composed of nine herbal plants, including and . The enumerated phytochemicals in the polyherbal extract include numerous bioactive compounds believed to confer therapeutic properties5. Moreover, it establishes a standardized approach for extraction and processing that utilizes decoction to concentrate the active compounds. This liquid is suitable for oral administration. A further benefit of traditional Ayurvedic medicines is the adaptability of the result6. This therapeutic approach typically yields an internally administered solution, but a tablet formulation is also feasible7. Nonetheless, applying these formulations is prevalent, and comprehension of the evidence supporting these interventions and the specific active components that underpin them needs to be improved, necessitating interdisciplinary collaboration to enhance our understanding of their therapeutic efficacy8.

The polyherbal decoction contains distinct phytochemical components. These compounds include alkaloids, flavonoids, tannins, glycosides, and amino acids that affect their anti-inflammatory, antioxidant, and gastroprotective properties9. Traditionally, polyherbal decoctions are recognized for enhancing the body's immune system, alleviating inflammation, and promoting overall wellness10. Nonetheless, empirical information substantiating these compounds' chemical composition, pharmacological properties, and prospective therapeutic applications is limited11. This deficiency in comprehension presents an opportunity to investigate and validate its efficacy in treating this condition using modern scientific methodologies12.

These investigations are being augmented by High-Performance Thin-Layer Chromatography (HPTLC) and Gas Chromatography–Mass Spectrometry (GC-MS), providing a robust foundation for the phytochemical examination of traditional treatments13, 14. These methodologies facilitate the identification of molecules with certain activities or attributes, enhancing the correlation between chemical structure and biological action. Furthermore, the comprehensive bioassay capabilities, including antioxidant, anti-inflammatory, and cytotoxicity assessments, provide insights into the formulation's pharmacology15. Integrating molecular docking and molecular dynamics simulations enhances comprehension of the binding affinities of essential bioactive chemicals and their target proteins, perhaps elucidating their pharmacological mechanisms16, 17.

The current study aims to provide a scientific foundation for the traditional applications of polyherbal decoction by analyzing its chemical composition and pharmacological effects. This study aims to demonstrate the efficacy of the formulated compounds through phytochemical analysis, HPTLC, nitric oxide inhibition, DNA binding, and molecular docking studies while exploring their potential applications in anti-inflammatory, antioxidant, and anticancer activities. The research seeks to elucidate the mechanism of the formulation by identifying molecular targets, including interleukin-8 (IL8), to discover novel treatments for inflammatory and gastrointestinal disorders18, 19.

This study further aims to integrate traditional herbal medical practices with contemporary scientific research to guarantee the use of safe and effective herbs in medicine. The planned extensive investigation would enhance the practical use of polyherbal decoction and further the identification of associated bioactive substances with potential therapeutic benefits. This aligns with the contemporary global trend in which several cultures incorporate traditional medicine into their healthcare systems, advocating for the use of natural products over synthetic pharmaceuticals.

MethodsDecoction preparation

The nine plants have been collected and authenticated. Using the cold percolated method, aqueous extract of all plants (, and ) has been prepared. All plants are collected, identified, and authenticated by Dr. K. Jeyaprakash, Botanist, Institute of Natural Science Research (Registered under the Indian Trusts Act), Devadanapatti - 625602, Tamil Nadu, India. Equal amounts of plant powder were weighed and soaked in distilled water for 3 days from time to time. After filtration, the extracts were concentrated and dehydrated to yield a semi-solid extract, which was stored at 4°C for further studies.

Preliminary Phytochemical Analysis

The polyherbal decoction was prepared using standard protocols. A dried semi-solid substance will be weighed to 100 mg and dissolved in 25 ml of distilled water for initial phytochemical screening10, 20. This analysis aims to identify the presence of several phytoconstituents, including alkaloids, quinones, polysaccharides, and terpenoids. Furthermore, the formulation will be tested to check for triterpenoids, glycosides, phytosterols, phenolics, bound compounds, steroids, flavonoids, amino acids, and tannins. These phytoconstituents will be identified using conventional methods specific to each group of compounds.

High-Performance Thin Layer Chromatography (HPTLC)

The chromatographic instrumentation utilized in this study was a CAMAG® Automatic TLC Sampler 4 (ATS 4). The plate development process was conducted in a glass chamber designed explicitly for chromatography13. The plate was evaluated using a TLC scanner 3, in conjunction with winCATS 4 software version 1. The samples were subsequently analyzed using High-Performance Thin Layer Chromatography (HPTLC) on aluminum-backed plates (10 x 10 cm) that were pre-coated with Merck silica gel 60 F254 (0.2 mm thickness). The plates were cleaned using methanol, heated to 60°C for five minutes, and subsequently, the sample was applied in four distinct quantities of ethyl acetate extract, namely 2 µL, 4 µL, 8 µL, and 12 µL, respectively. The samples were used as bands with a width of 8 mm at various positions on the plates, precisely 11 mm from the bottom and 15 mm from the side edges, using a Camag Linomat 5 semi-automatic applicator. The bands were desiccated by concurrently passing a nitrogen stream. The plate development was conducted using the ascending mode in a twin-trough glass chamber measuring 10 × 10 cm. The mobile phase consisted of n-hexane, Ethyl acetate, methanol, and formic acid in the 60:40:2.5:2.5 (v/v/v/v) ratio. The plates were subjected to densitometric scanning using a Camag TLC scanner three equipped with winCATS software, version 1.4.8, under UV light at a wavelength of 254 nm. The scanning process was conducted using a slit width of 5 mm by 0.45 mm, with a velocity of 20 mm/s and a data resolution of 0.121. The Rf values of the sample were measured, and photographs were captured using visible light and UV light at 254 and 366 nm wavelengths in the CAMAG REPROSTAR 3 photo documentation chamber. The peak counts, heights, peak areas, peak displays, and densitograms were acquired and examined using winCATS software.

Gas Chromatography/Mass Spectrometry (GC/MS)

A fraction of the identified polyherbal decoction was purchased from a renowned Ayurvedic store in Chennai. The sample was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) as per established protocols Gas Chromatography-Mass Spectrometry (GC–MS) BT - Encyclopedia of Geochemistry14. The study utilized an Agilent GC-MS system consisting of a G3440A gas chromatograph and a 7890A mass spectrometer, namely the 7000 Triple Quad GCMS model, equipped with a mass spectrometry detector. 100 µL of polyherbal decoction was combined with 1 mL of an ethanol solvent using a vortex stirrer for 10 seconds to prepare the sample. The transparent extract was obtained using this procedure, and the sample was then processed for gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS analysis was conducted using a DB-5MS column with dimensions of 30 mm × 0.25 mm ID × 0.25 µm. The column consists of 5% phenyl and 95% methyl polysiloxane. The study used electron impact mode at an energy level of 70 electron volts (eV). Helium, with a purity of 99.999%, was used as the carrier gas at a flow rate of 1 mL/min. The injector, auxiliary, and ion source temperature settings were 280°C, 290°C, and 280°C, respectively. The oven temperature is programmed to start at 50°C and remain constant for 1.0 minutes. Then, the temperature increases at a rate of 40°C per minute until it reaches 170°C, which remains constant for 4.0 minutes. Finally, the temperature is increased at a rate of 10°C per minute until it reaches 310°C, which remains constant for 10 minutes. A mass range of 45 to 450 Da was surveyed, and the entire duration of the run was 32.02 minutes. The compounds were characterized by comparing them to the reference spectra found in the NIST and WILEY GC-MS libraries22.

ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) radical scavenging assay The ABTS radical cation decolorization test measured the antioxidant activity23. ABTS radical cations were produced by combining equal volumes of a 7 mmol/L ABTS solution with a 2 mmol/L solution of sodium metabisulfite (NaSO) and potassium persulfate (KSO) at a concentration of 45 mmol/L. Subsequently, the mixture was placed in a dark environment at room temperature for 12 hours before utilization. The ABTS solution that was obtained was subsequently diluted with ethanol. The test samples were generated at concentrations ranging from 5 to 320 μg/mL, and the standard, ascorbic acid, was also synthesized at concentrations ranging from 5 to 320 μg/mL via serial dilution. The reaction solution is combined with 2 ml of the diluted ABTS solution. Once the reaction mixtures were created, they were allowed to sit at room temperature for 6 minutes before measuring the absorbance levels of the solutions using an ultraviolet-visible spectrophotometer at a wavelength of 734 nm. The experiment was conducted three times. The percentage of the radical scavenging effect of the samples was assessed using the formula employed to measure the ABTS radical scavenging activity. ABTS radical scavenging effect (%) = [(A - A)/A] ×100 Where A is the absorbance of control; A is the absorbance of test.

Ferric Reducing Antioxidant Potential Assay

The spectrophotometric method of Benzie & Strain, 199924 was used to assess the antioxidant activity of polyherbal decoction. This activity involves the conversion of Fe³⁺-TPTZ (colorless) to Fe²⁺-tripyridyltriazine (blue-colored complex) by donating an electron at low pH. The reaction was monitored using a spectrophotometer set at a wavelength of 593 nm. The Ferric Reducing Antioxidant Power (FRAP) reagent was prepared by combining 300 mM acetate buffer, TPTZ (10 mM) in 40 mM HCl, and 20 mM FeCl₃·6H₂O in a 10:1:1 ratio. The mixture was incubated at 37°C. A freshly manufactured FRAP reagent (3.995 mL) was prepared by combining it with the test samples and standard ascorbic acid at varying concentrations (5-320 μg/mL). The mixtures were incubated for 30 minutes at 37°C. During this time, the Fe³⁺-TPTZ complex underwent reduction, resulting in the creation of Fe²⁺ and the appearance of vivid blue color. The absorbance of this compound was determined at a wavelength of 593 nm using a reagent blank. The reagent blank was made by combining 3.995 mL of FRAP reagent with 5 μL of distilled water. The assessments were conducted thrice.

Protein Denaturation Assay

The anti-inflammatory efficacy of polyherbal decoction was assessed using a modified version of the protein denaturation method25. The diclofenac sodium medication was prepared using DMSO and diluted with a phosphate buffer (0.2 M, pH 7.4). The final concentration of DMSO in all solutions was kept below 2.5%. The drug was diluted in solutions of 4 mL with concentrations of 5, 10, 20, 40, 80, 160, and 320 μg/mL. These diluted solutions were then added to 1 mL of a 1 mM albumin solution in phosphate buffer. The mixture was left undisturbed for 15 minutes at a temperature of 37°C. The denaturation process was done by subjecting the reaction mixture to heat at 60°C for 15 minutes in a water bath. The turbidity of the samples was measured at a wavelength of 660 nm after they were cooled. The same process was also employed for the standard medication, and its level of cloudiness was recorded. The percentage inhibition of protein denaturation was calculated by comparing it to a control solution that did not contain the medication25.

Membrane Stabilisation Assay

Five milliliters of fresh whole human blood were drawn from healthy adult volunteers following informed consent, placed into a heparinized tube, and aliquoted into centrifuge tubes. The blood was spun at 3000 rpm for 10 minutes, and the pellets were washed thrice with normal saline at the same volume. The study protocol was approved by the Institutional Review Board (or Ethics Committee) of Bharath Institute of Higher Education and Research, Chennai (Protocol code BIHER/BSCI/IHEC/2021/15 dated 27.11.2021). The volume of the blood was then measured, and the blood was diluted to a 40% v/v suspension in an isotonic solution (10 mM sodium phosphate buffer)26. For the assay, a 40% RBC suspension was prepared by mixing 1 mL of packed RBCs with 1.5 mL of normal saline. Then, 1 mL of the suspension was incubated with varying concentrations of the polyherbal decoction, ranging from 50 to 1600 μg/mL, and the standard drug Diclofenac sodium. The control consisted of 1 mL of RBC suspension mixed with 1 mL of isotonic saline without the addition of any test substances. The reaction mixtures were incubated in a water bath at 56°C for 30 minutes. After incubation, the tubes were allowed to cool to room temperature and then centrifuged at 2500 rpm for 5 minutes. The absorbance of the supernatant was then read at 560 nm. Percent membrane stabilization activity was determined using the following equation: % Inhibition of Haemolysis = (OD of control - OD of test)/OD of control × 100.

Cell Culture and Cytotoxicity Assay

The antitumor properties of polyherbal decoction were assessed for cytotoxicity using an MTT assay on the A549 human lung carcinoma cell line. A549 cells were sourced from NCCS Pune, India, and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Genetix Biotech, India), penicillin/streptomycin (Sigma) at 37°C in a humidified atmosphere containing 5% CO. Human cells were plated at a density of 4,000 cells per well in 96-well plates and allowed to grow for 24 hours27. Polyherbal decoctions were dissolved in phosphate-buffered saline (PBS) to prepare 10 mg/mL stock solutions, and the final concentrations ranged from 1 to 1000 µg/mL. The treated cells were incubated at 37°C for 48 hours, and then 50 µL of 5 mg/mL MTT solution was added to each well and further incubated for 3 hours. Subsequently, the medium was removed from the wells, and the formed formazan crystals were dissolved in 100 µL DMSO. The absorbance was then taken at 570 nm using a Synergy HT microplate reader. Percentages of cell survival were also computed, and dose-response curves were plotted to obtain GI values. Microscopic examination of cell morphology was done at the end of 48 hours of treatment, and the pictures were taken to compare morphological alterations.

Statistical Analysis

Data are consistently presented as the average ± the standard error of the mean (SEM), allowing readers to assess the precision of the average. A one-way analysis of variance (ANOVA) was employed to determine if significant differences existed among the groups under various experimental settings. Dunnett’s multiple-comparison test was used after ANOVA to provide specific comparisons between each experimental group and the control group, reducing the likelihood of Type I errors by appropriate adjustment for multiple testing. Calculating 95% confidence intervals enables the estimation of the range within which the actual population parameters are likely to lie. Selecting a P-value threshold of < 0.05 assured that the observed mean differences were likely not due to chance. Arriving at these conclusions through a diverse array of evidence enhances their credibility and comprehensibility. All experimental assays were performed in triplicate (n = 3) to ensure reproducibility and statistical reliability. The sample sizes were selected based on preliminary studies and established protocols in similar in vitro experimental models. This approach strikes a balance between practical feasibility and the need for robust statistical analysis. Results are reported as mean ± standard error of the mean (SEM), and all assays were repeated independently to confirm consistency.

Molecular Docking

The extract's chemical compounds identified through GCMS analysis were evaluated using molecular docking techniques to determine their interaction with the Interleukin 8 protein target associated with irritable bowel syndrome28. The crystal structure of the target protein (PDB: 3IL8) indicates its role in the host defense system as a chemotactic factor29. Autodock Vina was chosen for the molecular docking process to model the protein-ligand complex30. The assessment of receptor-ligand binding affinities was organized and presented in a table to identify active compounds.

Molecular Dynamics Simulations

A molecular dynamics investigation was conducted on a protein-phytocompound combination. Molecular identification was performed by capturing frames throughout the trajectory creation using the GROMACS 2019 program31. Topologies for small molecules were generated with the PRODRG2 server, while the SPC water model and GROMOS96 43a1 force field were employed to define the protein topologies32. The cubic box enclosure facilitated solvation and energy minimization for the constructed complex system. To neutralize the solution, sodium (Na) and chloride ions were introduced to the receptor-ligand complex system. Energy minimization was performed utilizing the steepest descent integrator. The temperature and pressure were maintained at 300 K and 1 atm, respectively, with a leap-frog integrator. All molecular dynamics simulations were conducted in NVT and NPT ensembles, with save frame intervals exceeding 50 ns.

Table 1

Phytochemical analysis of polyherbal decoction showing the presence (+) or absence (-) of various bioactive compounds

1

Alkaloids

+

2

Quinones

-

3

Carbohydrate

+

4

Terpenoids

-

5

Triterpenoids

-

6

Glycosides

+

7

Phytosterols

+

8

Phenolic compounds

-

9

Steroids

-

10

Flavonoids

+

11

Amino acids

+

12

Tannins

+

ResultsPreliminary Phytochemical Screening

The phytochemical analysis of polyherbal decoction revealed the presence of multiple bioactive chemicals that may contribute to the drug's therapeutic effects. Based on the positive results obtained in the corresponding tests, the formulation contained alkaloids, carbohydrates, glycosides, phytosterols, flavonoids, amino acids, and tannins (Table 1 ). Alkaloids have a wide range of pharmacological actions and have been discovered to have anti-inflammatory and antibacterial properties33. Carbohydrates possess energy-providing and immune-regulating qualities, whereas glycosides are linked to cures for heart-related issues and have anticancer action34, 35.

Phytosterols, which are linked to cholesterol regulation, play a role in enhancing heart health. The flavonoids in the polyherbal decoction have been recognized for their antioxidant, anti-inflammatory, and antiviral characteristics36. Proteins, composed of amino acids, may play a role in tissue regeneration and metabolic processes37. The plant also contains tannins, which have been found to possess astringent, antibacterial, and anti-inflammatory properties38. However, examining quinones, terpenoids, triterpenoids, phenolic compounds, and steroids reveals that these compounds do not play a role in bioactivity. The results unequivocally support the traditional use of polyherbal decoctions for medicinal purposes and contribute to our understanding of their potential pharmacological effects. Further research is needed to thoroughly clarify the pharmacological effects and therapeutic efficacy of these substances in humans.

Figure 1

HPTLC analysis of polyherbal decoction. (A) Chromatographic separation of phytochemicals visualized at 254 nm (left) and 366 nm (right) showing distinct bands. (B) 3D densitometric profile of tracks scanned at 254 nm.

HPTLC Profile

The High-Performance Thin-Layer Chromatography (HPTLC) analysis revealed twelve distinct peaks in the sample when exposed to ultra-violet (UV) light at 254 nm and 366 nm39, as shown in Figure 1. This indicates that the sample hosts numerous compounds. Two prominent peaks were found, indicating the presence of two main chemicals in the sample. Hence, our findings demonstrate the diverse range of phytochemicals present in the sample and highlight the need for further investigation to identify the bioactive molecules. The presence of a peak indicated no discernible distinction between the two samples. The data suggests that a 6% portion of the overall curve area could include Compound A despite its minor component. However, this small fraction could still hold significant importance. Simultaneously, a prominent peak at an Rf value of 0.68 corresponds to a representation of 44. The results obtained a reliability of 48% for the entire area, suggesting that Compound B is the primary component of the sample. The significant abundance of Compound B implies that it likely accounted for the majority, if not the entirety, of the biological or therapeutic impact observed in the sample. The discovery of these compounds could be crucial in establishing the sample's efficacy and potential application in a more robust formulation.

Figure 2

A GC-MS chromatogram of the polyherbal decoction shows total ion current (TIC) across retention times. Prominent peaks are observed at 5.726 min, 9.707 min, 16.090 min, 20.378 min, 24.211 min, 25.764 min, and 28.113 min, indicating the presence of major bioactive compounds in the sample. The data suggests a complex mixture of volatile components consistent with the diverse phytochemical profile of the traditional Ayurvedic formulation.

Table 2

It presents the retention values, types of possible compounds, their molecular formulae, molecular mass, peak area, and the medicinal roles of each compound, as shown in the GC MS profile of polyherbal decoction

5.68

Benzoic acid

C7H6O2

122

37.91

Acidifier, Arachidonic acid Inhibitor, Increase Aromatic Amino acid decarboxylase activity, Inhibit production of uric acid, Urine acidifier

15.73

11-Octadecenoic acid, methyl ester

C19H36O2

296.3

0.92

Arachidonic acid Inhibitor, Increases Aromatic Amino acid decarboxylase activity, Inhibits production of uric acid, Urine acidifier, Catechol O Methyl transferase inhibitor, Methyl donor, Methyl guanidine inhibitor, Acidifier

16.09

17-Octadecynoic acid

C18H32O2

280.2

0.59

Arachidonic acid Inhibitor, Increases Aromatic Amino acid decarboxylase activity, Inhibit production of uric acid, Urine acidifier, Catechol O Methyl transferase inhibitor, Methyl donor, Methyl guanidine inhibitor, Acidifier

17.27

2-Cyclohexyl-2,5-cyclohexadiene-1,4-dione, 4-oxime

C12H15NO2

205.1

1.11