Chemico-pharmacological evaluation of the methanolic leaf extract of Catharanthus ovalis: GC–MS/MS, in vivo, in vitro, and in silico approaches

Introduction: Natural plant-based medicines have gained popularity recently as a major source of inventive, risk-free, and more potent secondary bioactive compounds with medicinal potential. Catharanthus ovalis is a perennial shrub containing various indole alkaloids cultivated extensively for local medical purposes. Methods: This research is conducted to identify the phytocompounds present in the leaves of C. ovalis and its central and peripheral analgesic, thrombolytic, and membrane-stabilizing activities through tail immersion, acetic acid-induced writhing, human blood clot lysis, and erythrocyte lysis by heat and hypotonic solution methods, respectively. Results and discussion: A total of 39 compounds were identified using GC–MS/MS techniques, including hexadecanoic acid, methyl ester (56.749%), methyl stearate (29.782%), carvacrol and its TBDMS derivative (12.586%), and 9-octadecenoic acid, methyl ester, (E)-] (9.297%) presented in high quantity. The highest tail immersion latency was observed for the 600 mg/kg extract of C. ovalis crude extract. Both 400 and 600 mg/kg doses of C. ovalis crude extract exhibited prominent peripheral analgesic activity. The maximum thrombolytic effect was observed by DCM soluble fraction extract by inhibiting 54.87% of the clot. However, the aqueous-soluble fraction of this extract manifested an excellent membrane-stabilizing effect by showing 73.98% and 87.51% hemolysis against heat- and hypotonic-induced hemolysis, respectively. Some of the compounds were identified as active agents against different receptors related to these diseases, which supported the findings of in vitro and in vivo tests. Conclusion: Further investigation needs to be conducted to specify and identify the exact mechanism of action of these compounds.


GRAPHICAL ABSTRACT 1 Introduction
Since ancient times, plants have been used as excellent sources of medicines to treat many illnesses.Recently, natural plant-based products have drawn attention as a key source of creative, safer, and more potent secondary bioactive metabolites with therapeutic potential.Nearly 80% of all pharmaceuticals are either wholly or partially derived from plants (Obonti et al., 2021).According to the World Health Organization (WHO), 80% of people utilize herbal medicines as part of their primary healthcare.Plant-based treatments are anticipated to make up to 25% of all drugs in developed nations such as the United States while making up to 80% of all drugs in rapidly growing nations such as India and China.Even in the modern period, plants are still a potential source of medication for serious illnesses such as cancer, oxidative stress, cancer, diarrhea, depression, fever, and thrombosis (Islam et al., 2022).It has been demonstrated that in vitro screening techniques can offer essential initial observations required to choose crude plant extracts with potentially beneficial features for additional chemical and pharmacological research (Mathekga and Meyer, 1998).
Catharanthus is a genus of flowering plants of the Apocynaceae (dogbane) family (Tolambiya and Mathur, 2016).It has glossy opposing leaves, and its stunning five-petaled flowers come in a range of pink, white, and purple hues and can be distinguished by its evergreen, herbaceous, or subshrubby habit (Mishra and Verma et al., 2017).Typically, there are eight species in this genus, seven of which are native to Madagascar, namely, Catharanthus coriaceus, Catharanthus lanceus, Catharanthus longifolius, C. ovalis, Catharanthus roseus, Catharanthus scitulus, and Catharanthus trichophyllus, and one of which, namely, Catharanthus pusillus, has been reported from India (Alam et al., 2017).Historically, the Caribbean native plant family Apocynaceae has been utilized for a variety of medical conditions; especially, European herbalists used plants of these families for diabetes and illnesses as diverse as headaches, as well as for wound healing (Kabesh et al., 2015).
However, the Catharanthus genus is well known for its diverse phytochemical composition, especially for the high concentrations of alkaloids, terpenoids, and flavonoids.Vinblastine and vincristine are two of the most noteworthy alkaloids and have received much attention for their powerful anticancer properties.In addition, several other bioactive substances have been discovered, contributing to a variety of pharmacological actions (Pham et al., 2020).Catharanthus roseus, the most common species of the Catharanthus genus rooted in traditional folklore, is revered for its diverse applications in treating burns, rheumatism, and menstrual disorders and its potential in managing hypertension, diabetes, and cancer and supporting uterine contractions (Chaturvedi et al., 2022;De et al., 2022).
Catharanthus ovalis Markgr., a perennial plant, typically grows in dry shrubland or desert biomes and can grow up to 40 cm in height (Tropical Plants Database, 2023).Although indigenous to southern Madagascar and Africa, it is cultivated extensively for local medical purposes.Historically, the plant's pure extract was consumed as a trial poison.Additionally, worms are expelled using an extract of the aerial sections.Indole alkaloids, including many bisindole alkaloids, are abundant in this species.On the other hand, it comprises several pharmacologically effective chemicals such as leurosine, vindoline, vindolinine, coronaridine, catharanthine, and vinblastine (Tropical Plants Database, 2023).Despite its recognized medicinal potency, there is a notable absence of comprehensive pharmacological research on this species, limiting our understanding of its full therapeutic potential and applications.The purpose of this investigation was to identify the phytochemicals present in the C. ovalis leaf tissue through GC-MS/MS techniques.Additionally, this study used in vivo and in vitro methodologies to examine several biological functions of the leaf extract.However, the in silico approach was adopted for the preliminary screening of the identified phytochemicals responsible for those biological activities.

Plant material
Catharanthus ovalis Markgr.was collected from Sylhet, Bangladesh, and was identified by the Bangladesh National Herbarium with an accession number of DACB 88049.The plant is prepared and preserved in the phytochemical research laboratory of the Department of Pharmacy at the State University of Bangladesh.

Preparation of extract and subsequent fractions
The C. ovalis leaf material was taken from the wild, shadedried, and turned into a powder using a mechanical grinder.The needed amount of leaf powder was weighed, transferred to a flask, totally submerged in methanol, incubated for 15 days, and then, filtered.Following that, a rotary evaporator was used to concentrate the filtered extract (Islam et al., 2020).The concentrated extract contains both polar and nonpolar plant material components, and 2 µL of the sample solution was used in GC-MS/MS for chemical analysis.
Solvent-solvent partitioning was conducted using the process described by VanWagenen et al. (1993).A measure of 5 g of the crude methanolic extract of C. ovalis (COCME) was dissolved in 10% aqueous methanol and then extracted sequentially with petroleum ether, dichloromethane, ethyl acetate, and finally, water to obtain four respective fractions: petroleum ether-soluble fraction (COPESF), dichloromethane-soluble fraction (CODMSF), ethyl acetate-soluble fraction (COEASF), and aqueous-soluble fraction (COASF).All the fractions were evaporated to dryness and were used for further analysis.

GC-MS/MS analysis and the identification of phytocompounds
A GCMS-TQ8040 GC-MS/MS (Shimadzu, Japan) system with an Elite-5MS (5% diphenyl/95% dimethyl polysiloxane) and a fused capillary column (30 0.25 m ID 0.25 m df) was used to conduct the GC-MS/MS analysis of the methanolic extract of C. ovalis.The electron ionization system employed for GC-MS/MS detection operated in an electron impact mode with an ionization energy of 70 eV.An electron ionization system with an ionization energy of 70 eV was run in an electron impact mode for GC-MS/MS detection.As a carrier gas, helium gas (99.999%) was used with an injection volume of 2 µL and a constant flow rate of 1 mL/min (a split ratio of 10:1).The oven temperature was programmed to start at 50 °C (isothermal for 1 min), increase by 15 °C/min to 200 °C, then 5 °C/min to 300 °C, and end with a 7 min isothermal at 300 °C.The injector temperature was kept at 250 °C.At a scan speed of 2000, mass spectra were collected at 70ev.The entire running time for the GC/MS was 36 min, and the solvent delay was 3.50 min.The spectrum was measured between 50 and 600 m/z.Comparing each component's average peak area to the total areas allowed us to determine the proportional percentage amount of each component.The identification of compounds using GC-MS spectra involved leveraging the National Institute of Standards and Technology (NIST) database, which contains approximately 62,000 patterns.The spectra of the unknown components were compared against both the known components stored in the NIST collection and the database's extensive collection of patterns.The NIST database, which contains more than 62,000 patterns, was used for the GC-MS/MS experiment.A comparison was made between the spectra of the unknown components and the spectrum of known components kept in the NIST collection.The components of the test materials' names, molecular weights, and structures were determined and listed in Table 1 (Sermakkani and Thangapandian, 2012).

In vitro test 2.4.1 In vitro thrombolytic activity
Streptokinase (SK) was used as the reference standard for the evaluation of the thrombolytic activity of crude methanolic extract (COCME), petroleum ether soluble fraction (COPESF), dichloromethane soluble fraction (CODMSF), ethyl acetate soluble fraction (COEASF), and aqueous-soluble fraction (COASF) of the leaf extract of C. ovalis using the technique developed by Prasad et al. (2006).
At first, six Eppendorf tubes were weighed, and then, 500 μL of the volunteers' venous blood was placed in each tube and incubated for 45 min at 37 °C.After the coagulation formed, the serum was removed from this tube.To confirm the weight of the clot, each tube was reweighed.Then, 100 μL of aforementioned fractions and standard streptokinase were correctly added to each Eppendorf tube.Each tube underwent a further 90-min incubation at 37 °C for coagulation.After incubation, the fluid was removed, and the tubes were weighed again to measure the weight change following clot disruption.The final percentage of clot lysis was determined by measuring the weight difference before and after the clot lysis process.
The ultimate percentage of clot lysis was calculated using the weight difference between before and after clot lysis.

% clot lysis
Weight of the lysis clot Weight of the clot before lysis × 100%.

In vitro membrane-stabilizing activity
The approach published in the work of Sikder et al. (2012) was used to assess the membrane-stabilizing ability of the plant samples.This method involves hemolysis brought on by heat and hypotonic solution.The blood was collected, and ethylenediaminetetraacetic acid was used as an anticoagulant.Then, the blood was washed with an isotonic solution (0.9% NaCl) and centrifuged for 10 min at 3,000 rpm speed.This washing was carried out three times, and finally, the stock erythrocyte was collected.

Hemolysis induced by a hypotonic solution
A hypotonic solution was used during the experimentation.The test material, including COCME, COPESF, CODMSF, COEASF, and COASF at a dose of 2.0 mg/mL and aspirin (standard) at a dose of 0.10 mg/mL, was mixed separately with 0.5 mL stock erythrocyte suspension, 4.5 mL hypotonic solution (0.3% NaCl), and 10 mM sodium phosphate buffer (pH 7.4).For the negative control group, water is used in place of the test material.The mixes underwent a 10min incubation at room temperature, followed by a 10-min centrifugation at 3,000 rpm.Finally, the supernatant's absorbance [optical density (OD)] was taken at 540 nm using a UV spectrophotometer.
The hemolysis percentage was calculated using the following equation: Here, OD1 is the optical density of the hypotonic-buffered saline solution alone (control group); OD2 is the optical density of the test sample in the hypotonic solution.

Hemolysis induced by heat
At first, 4.5 mL of isotonic solution and 10 mM sodium phosphate buffer solution and the standard aspirin at a dose of 0.10 mg/mL or the abovementioned samples at a dose of 1 mg/mL were poured into two sets of different centrifugal tubes.Then, 30 µL of stock erythrocytes were mixed with each of the tubes.One set of tubes was incubated in a water bath at 54 °C for 20 min.The second pair was kept in an ice bath at 0 °C-50 °C for the same time.After that, these mixtures were centrifuged for 3 min at 3,000 rpm speed, and then, UV spectrophotometric absorbance was taken at 540 nm.
The hemolysis percentage was calculated using the following equation: Here, OD 1 is the test sample unheated, OD 2 is test sample heated, and OD 3 is the control sample heated.

In vivo test 2.5.1 Test animal
Swiss albino mice aged 4-5 weeks of either sex were procured from the International Center for Diarrheal Disease and Research in Bangladesh for use in in vivo biological experiments (ICDDR, B).They received free access to rodent chow and water while being housed at a constant room temperature of 24 °C ± 2 °C and relative humidity levels of 60%-70% for 12 h of light and darkness.However, food was withheld 12 h prior to the experiment and throughout its duration.The care and use of experimental animal guidance were followed in conducting the studies.The institution's ethics committee gave its approval to the procedures for using animals in the tests.

Study design for the in vivo test
The experimental models consisted of five groups of Swiss albino mice, with each group containing four mice, and the assignment to the groups was performed randomly.For the various experimental models, five groups of three Swiss albino mice were randomly assigned, where each group contained four mice.The negative control group, serving as the first group, received 10 mL/kg of distilled water.The first group acted as the negative control and received 10 mL/kg of distilled water.The second group acted as the experiment's positive control and was administered normal medications.The third, fourth, and fifth groups received 200, 400, and 600 mg/kg b.w dose of COCME, respectively.

In vivo central analgesic activity
The tail immersion method described by Ezeja et al. ( 2011) was used to determine the central analgesic activity of COCME.Diclofenac sodium (50 mg/kg bw) solution was administrated as standard.COCME (200, 400, and 600 mg/kg bw) for test groups and distilled water for the negative control group were administrated orally.In order to conduct the test, 1-2 cm of the mouse's tail was dipped into a water bath filled with warm water that was kept at a constant temperature of 55 °C.The mouse's tail deflection time was timed at 0, 30, 60, and 90 min after the treatments were administered.
The following equation was used to calculate the % time elongation, which was then compared to the reference value to assess the central analgesic action.
The central analgesic action of the group increases with group elongation %.

% time elongation
Here, Ttest is the average time of tail deflection in the test group, and Tcontrol is the average time of tail deflection in the control group.

In vivo peripheral analgesic activity
To ascertain the peripheral analgesic effect of C. ovalis in albino mice, the acetic acid-induced writhing test was carried out as previously reported (Satyanarayana et al., 2004).Diclofenac sodium (50 mg/kg bw) solution was injected intraperitoneally as standard.Test groups were given three different doses including 200, 400, and 600 mg/kg bw of COCME, and the negative control group was given only distilled water.After 30 min of sample administration, glacial acetic acid (0.1 mL/30 mg bw) was injected through the intraperitoneal route.For each group of mice, the number of abdominal constrictions, or writhes, was measured from 5 min after the injection of acetic acid until 10 min later, and the percentage inhibition of writhing was calculated using the following formula to assess the peripheral analgesic activity: Here, Wtest refers to the average number of writhing in the test group, and Wcontrol refers to that in the control group.

In silico study 2.6.1 Docking software
The molecules were subjected to computational docking analysis, employing widely recognized software applications such as PyRx, PyMOL 2.3, Discovery Studio 4.5, and Swiss-PdbViewer, to explore their molecular interactions and binding affinities in a virtual environment (Hasnat et al., 2023).

Molecular docking: ligand preparation
The structures of 39 isolated compounds of the C. ovalis leaf extract and aspirin (PubChem CID-2,244), diclofenac sodium (PubChem CID-3,033), and streptokinase (PubChem CID-9815560) were downloaded from the PubChem database and are presented in Table 1 (https://pubchem.ncbi.nlm.nih.gov/).In order to obtain the best possible hit rate against the aforementioned targets, the ligands were downloaded in the 3DSDF format.These ligands, along with their PubChem CIDs, were serially loaded in the Discovery Studio 4.5.It should be mentioned that the Pm6 semiempirical technique was used to optimize all phytochemicals in order to improve docking accuracy (Bikadi and Hazai, 2009).

Molecular docking: protein preparation
Three-dimensional crystal structures, including the mu-opioid receptor (PDB ID: 5C1M) for central analgesic activity (Ahmed et al., n.d.), cyclooxygenase-2 (COX-2) [PDB ID: 1CX2] for peripheral analgesic activity (Muhammad et al., 2015), tumor necrosis factor alpha (TNF-α) [PDB ID: 2AZ5] for membranestabilizing activity (Kumar et al., 2021), and tissue plasminogen activator (TPA) [PDB ID: 1A5H] for thrombolytic activity (Emon et al., n.d.), were collected from the RCBS Protein Data Bank (https://www.rcsb.org/structure) in the PDB format.Using Discovery Studio 2020, all of the water and the heteroatoms were taken out of the proteins.Then, using the Swiss-PdbViewer's energy reduction software, all the biomolecules were organized by adding nonpolar hydrogen atoms and kept in their lowest energy state for future research.

Ligand-protein interaction
To forecast potential binding profiles of isolated compounds with their binding affinities to the target molecules, the present computer-aided ligand-protein interaction was drawn (Bikadi and Hazai, 2009).Regarding the process of linking molecular drugs to proteins, for this molecular drug-protein linking procedure, highly sophisticated PyRxAutodock Vina was used, and semi-flexible modeling was used for the molecular docking.Initially, the protein was loaded and prepared to interact with the target macromolecule.To ensure specific ligand binding to the target, we selected amino acids with their respective IDs from the literature.The protein was first loaded and formatted to the target macromolecule, and the literature-based amino acids with their ID were chosen to ensure target-specific ligand binding.
Next, in order to find the most optimal binding interactions with the selected macromolecules, all the ligands' PDB files were imported and then converted to the pdbqt format using the Open Babel tool within the PyRxAutoDock Vina software.Subsequently, the minimized ligand structures were employed for docking simulations.
Second, to match the best optimal hit during the docking against these selected macromolecules, all the PDB files of the ligands were imported and afterward minimized into the pdbqt format with the Open Babel tool in the PyRxAutoDock Vina software.
Upon docking, the remaining parameters were set to their default values.Subsequently, using AutoDock Vina (version 1.1.2),computer-based-aided molecular docking of the ligand compounds was carried out while maintaining all essential conditions default (Ahmed et al., 2021).Finally, BIOVIA Discovery Studio version 4.5 was used to conceptualize all docking analyses for extrapolating and concluding the best-fitted figures using 2D and 3D configurations.

Statistical analysis
The data were presented as a standard error mean (SEM).Statistical analyses using one-way ANOVA were conducted followed by Dunnett's multiple comparison tests.The observed values were compared to the control group and were considered statistically significant at p < 0.05, p < 0.01, and p < 0.001 (Miah et al., 2018).

GC-MS/MS analysis
The sample produced 39 peaks on the GC-MS/MS chromatogram (Figure 1), indicating 39 different phytochemical components.Following a comparison of the mass spectra of the constituents in the NIST library with those of the picks, 39 phytocompounds were defined and identified (Table 1).

Thrombolytic activity
In this investigation, the CODMSF exhibited an extremely strong thrombolytic impact by inhibiting 54.87% of the clot, which is close to the standard streptokinase (66.66%) (Figure 2).Additionally, COCME and COAQSF fractions demonstrated noteworthy anti-thrombosis effects through suppressing clots by 37.65% and 45.32%, respectively, while the control group only showed 7.06% clot lysis.This result signifies that there is a huge opportunity to develop new thrombolytic drugs from C. ovalis.

Membrane-stabilizing activity
Our study demonstrated that the COAQSF has manifested remarkable membrane-stabilizing activities exceeding aspirin (standard) activities with twice its value.Figure 3 illustrates that COAQSF inhibited 73.98% and 87.51% hemolysis against heat and hypotonic solution-induced hemolysis, respectively, while the values were 3.08% and 22.26%, respectively, for the standard streptokinase.However, CODMSF exhibited prominent activity followed by COEASF, where the inhibition of hemolysis was measured at 27.27% and 17.67% for COSMSF and 13.81% and 12.17% for COEASF against heat-and hypotonic solution-induced hemolysis, respectively.This finding enhanced the probability of developing new remedies against inflammation.

Central analgesic activity
By extending the experimental mice's tail immersion period, the various dosages of the crude methanolic extract of C. ovalis (200, 400, and 600 mg/kg) demonstrated considerable central analgesic efficacy.A dose-and time-dependent increase in the activity was observed.The highest activity was observed for 600 mg/kg extract which increased the tail emerged time to approximately 139.75 ± 0.23, 222.34 ± 0.07, and 381.14% ± 0.55%, respectively, after 30, 60, and 90 min of dose ingestion (Table 2).

Peripheral analgesic activity
A remarkable peripheral analgesic activity was observed for different dosages of the COCME, especially for the dose of 400 mg/kg and 600 mg/kg.In comparison to the control (34.5 mean writhings), an average of 16.5 mean writhings was witnessed in the 200 mg/kg dose group, and 9.5 mean writhings were noted in the 400 and 600 mg/kg extract groups (Table 3).This indicates that the 200, 400, and 600 mg/kg COCMEs reduced writhing by approximately 32.65 ± 1.19, 61.22 ± 1.04, and 61.22% ± 1.85% , respectively.All of the data were significantly justified.
When compared to regular aspirin (−5.3 kcal/mol), C30 had a binding affinity for TNF-α (PDB ID: 2AZ5) of −9.3 kcal/mol.C7, C27, C28, and C39 all had binding affinities of −7.5 kcal/mol, whereas C34 had a binding affinity of −7.9 kcal/mol.However, with binding affinities ranging from −6.5 to −5.8 kcal/mol, C3, C9, C21, C26, C29, and C36 scored higher affinities compared to aspirin.In the case of active amino acid residue, C30 was found to bind against TYR 59 TYR 119 of the A chain and TYR 59 of the B chain.Similarly, C39 bound to TYR 59 TYR 119 of the A chain and LEU 57 of the B chain, whereas aspirin bound to HIS 57, TYR 99, ALA 190,CYS 191,GLN 192,GLY 193,TRP 215, and GLY 216 of the A chain (Figure 6).

Discussion
The best place to find novel bioactive compounds with new treatments is in medicinal plants.As a result, plant-based natural remedies are widely used in developing nations and given special attention because of their multiple protective benefits and beneficial effects on human health.Approximately 80% of people use traditional medicines, even in underdeveloped nations (Alam et al., 2020a).Oils contained by plant extract are typically intricate blends of vital secondary metabolites that have been extracted from plants, animals, and microorganisms.These mixes contain between 10 and 60 ingredients in varying concentrations, but often just 2-4 main molecules are responsible for the biological features (Rahman et al., 2013).The goal of the current research was to identify the chemical components and assess their biological and pharmacological potential in order to understand the significance of the methanolic crude extract of C. ovalis.The study will provide an update, confirm the value of aromatherapy, and add a new bearing on biopotency.
The GC-MS/MS analysis was carried out to identify the various volatile matter components found in the methanolic crude extract of C. ovalis leaves, such as alcohols, acids, amides, amines, esters, and hydrocarbons, both in quantitative and qualitative ways.A quantitative measurement of each component's quantity can be found in peak heights or regions beneath the peaks.This study identified 39 phytocompounds from the methanolic crude extract of leaves of C. ovalis while the maximum peak was acquired for hexadecanoic acid [methyl ester], which is approximately 56.749%, indicating the presence of a large quantity of this compound in this extract.
Hexadecanoic acid has been reported for potential antioxidant activity as well as anticancer potential against human colon cancer cells (Bharath et al., 2021).In addition, the compound exhibited  significant antibacterial activity against several antibiotic-resistant bacteria including Staphylococcus aureus W35, Pseudomonas aeruginosa D31, Klebsiella pneumoniae DF30, and K. pneumoniae B40 (Shaaban et al., 2021).Furthermore, methyl ester of hexadecanoic acid isolated from Annona muricata Linn.has been found promising antifungal potential against Alternaria solani, Aspergillus erythrocephalus, and Aspergillus albicans (Abubacker and Deepalakshmi, 2013).However, the presence of a significant The best critical interactions in the binding pocket for the chosen ligands and receptors are represented in 3D and 2D, where A to G represent the interaction of C7, C28, C30, C34, C36, C39, and standard (diclofenac sodium) with the mu-opioid receptor, respectively.
amount of hexadecanoic acid in C. ovalis leaf extract increases the possibility that it has natural cytotoxic, antibacterial, and antifungal properties.
Our investigation revealed a substantial presence of methyl stearate, constituting approximately 29.782%, known for its medicinal properties.Notably, as a bioactive compound identified in the fermentation broth, methyl stearate plays a pivotal role in inhibiting nematode infection.Its efficacy is demonstrated through actions such as reducing egg hatching, repelling J2s from plant roots, and modulating key parasitic nematode genes (Mi-flp-18 and 16D10).This compound shows promise in effectively controlling Meloidogyne incognita.Additionally, its positive influence on enhancing banana plant growth at lower concentrations suggests its potential as an environmentally friendly approach to nematode control.This presents a viable alternative to conventional pesticides, contributing to sustainable agricultural practices (Lu et al., 2020).
Blood flow in the circulatory system is impeded if a thrombus forms inside blood arteries, which can lead to a number of fetal cardiovascular illnesses including hypertension, stroke, and anoxia.Thrombolites are substances that prevent thrombus formation, hence managing and treating embryonic cardiovascular disorders (Islam et al., 2019).Several studies have been conducted in the past using medicinal plants from Bangladesh to identify natural chemicals having anti-thrombolytic potential (Rahman et al., 2013).The research discovered that the dichloromethane (DCM) fraction of the methanolic leaf extract from C. ovalis exhibited notable thrombolytic activity.Moreover, both the crude methanolic extract and the aqueous fraction demonstrated encouraging properties in dissolving blood clots, which increases the potential for developing novel preparations derived from this plant to mitigate cardiovascular diseases.
Inflammation causes lysosome lysis, which releases enzymes that cause different types of tissue damage.Inflammation causes lysosome lysis, which releases enzymes that cause different types of tissue damage.When an RBC is exposed to adverse circumstances or substances such as hypotonic medium or heat, the membranes break due to hemolysis and hemoglobin oxidation.In times of enhanced permeability brought on by inflammatory mediators, the stability of the membrane limits the passage of proteins from the blood and fluids into the RBC (Kosala et al., 2018).Thus, by stabilizing RBC induced by both hypotonic solution as well as heat, the aqueous fractions illustrated extremely potent anti-inflammatory activity compared to the standard aspirin.Furthermore, the crude methanolic extract from the leaves of C. ovalis demonstrated a The best critical interactions in the binding pocket for the chosen ligands and receptors are represented in 3D and 2D, where A to H represent the interaction of C7, C15, C21, C22, C27, C36, C39, and standard (diclofenac sodium) with the COX-2 receptor, respectively.
remarkable ability to prevent hemolysis, approaching the efficacy of the standard.A study represented that hexadecanoic acid, as evidenced by its stable binding within the active site of phospholipase A2 (PLA2), demonstrates a potential anti-inflammatory effect by impeding substrate entry and hindering catalytic residues.Understanding its interaction with PLA2 may contribute to the development of drugs targeting chronic inflammatory conditions such as rheumatoid arthritis and asthma The best critical interactions in the binding pocket for the chosen ligands and receptors are represented in 3D and 2D, where A to M represent the interaction of C3, C7, C9, C21, C26, C27, C28, C29, C30, C34, C36, C39, and standard (aspirin) with the TNF-α receptor, respectively.
Frontiers in Pharmacology frontiersin.org13 (Aparna et al., 2012).So, our finding suggests that the presence of high amount of hexadecanoic acid is the reason for the extract potential membrane-stabilizing effect.
Using the tail immersion test, we assessed the central analgesic ability of the crude extract of C. ovalis.This procedure results in supra-spinal pain that is centrally mediated.While it is generally established that acetic acid somehow triggers the release of endogenous pain mediators, activating the pain-sensing neurons (Arslan and Bektas, 2010), in this study, the peripheral analgesic activity of the crude methanolic extract from the leaves of C. ovalis was determined by inducing writhing on mice with acetic acid.
This study unveiled a statistically significant and noteworthy central analgesic effect from the crude methanol leaf extract of C. ovalis, which was both dose-and time-dependent.Interestingly, the moderate dose (400 mg/kg) exhibited the most potent peripheral analgesic activity, with statistically significant results, followed by the highest dose (600 mg/kg).This finding could serve as an initial avenue for further investigation aimed at uncovering more precise insights into the plant's pain-relieving properties.
To better understand the biological roles of substances from nature and to estimate ligand-receptor interactions, molecular docking studies were widely used.It offers further details on potential methods of action and binding style in the binding sites of certain proteins (Alam et al., 2020b).
As a matter of fact, a molecular docking investigation was carried out to clarify and validate the experimental findings from biological research.This technique confirms the experimental results.In order to find the central and peripheral analgesic properties, the identified compounds docked against Mu (PDB ID: 5C1M) and COX-2 (PDB ID: 1CX2) receptors, respectively, while TNF-α (PDB ID: 2AZ5) and TPA (PDB ID: 1A5H) receptors were selected for screening the antiinflammatory and thrombolytic activities of identified compounds from the crude methanolic extract of leaves of C. ovalis.
FIGURE 1 GC-MS/MS chromatograph of the crude methanolic extract of leaves of C. ovalis.

FIGURE 2
FIGURE 2Thrombolytic effect of different fractions of the C. ovalis leaf extract.Each value is expressed as mean percentage (n = 3), *p < 0.05, **p < 0.01, and ***p < 0.001, and compared with the control group.

FIGURE 3
FIGURE 3Membrane-stabilizing effect of different fractions of the C. ovalis leaf extract.Each value is expressed as mean percentage (n = 3), *p < 0.05, **p < 0.01, and ***p < 0.001, and compared with the standard group.

TABLE 1 A
total number of 39 compounds identified by GC-MS/MS analysis from the leaf extract of C. ovalis with their molecular weight and PubChem ID.

TABLE 2
Central analgesic effect of different doses of the crude methanolic extract of the leaves of C. ovalis in the mice model.Each value is expressed as mean ± SEM (n = 4).*p<0.05, **p < 0.01, ***p < 0.001 and compared with the control group (only distilled water).TABLE3Peripheral analgesic effect of different doses of the crude methanolic extract of the leaves of C. ovalis in the mice model.Each value is expressed as mean ± SEM (n = 4), *p < 0.05, **p < 0.01, and ***p < 0.001, and compared with the control group (only distilled water).Binding affinities of the identified compounds and standards against four receptors representing central and peripheral analgesic, membranestabilizing, and thrombolytic activities.

TABLE 4 (
Continued) Binding affinities of the identified compounds and standards against four receptors representing central and peripheral analgesic, membrane-stabilizing, and thrombolytic activities.

TABLE 6
Metabolism and excretion profile of the selected compounds which showed the best interaction against these receptors.

TABLE 7
Toxicology and drug-likeliness profile of the selected compounds which showed the best interaction against these receptors.