Biological and in silico investigation of isolated novel bioactive compound from Conocarpus lancifolius

1 Introduction

Medicinal plants are the source of large number of secondary metabolites such as anthocyanins, terpenoids, phenolics, flavonoids, polyphenols, alkaloids and carotenoids in a different concentration and shield humans for development of various diseases. Plant extracts are used in pharmaceuticals due to the presence of these bioactive compounds. Natural products as extract or pure compound provide opportunities for drug discovery and development (Policegoudra et al., 2012). The focal of researchers on the development of medicines from natural products because synthetic medicines have side effects and contraindications (Petrovska, B. B., 2012). The medicinally important bioactive compounds are isolated from plant extract for various therapeutic purposes (M. A., 1999). Despite huge research on natural medicinal products, some phytochemicals are not fully identified even the mechanism of action of phytochemicals is also not completely understood. However, the main target of researchers is to isolate bioactive compounds for therapeutic purposes (Irshad et al., 2021). Many medicinal plants have cytotoxicity, antioxidant, anti-diabetic, anticancer, anti-inflammatory and antibacterial activities (Abdel-Hameed et al., 2012; Afsheen and Jahan, 2013; Alam et al., 2013; Greenwell and Rahman, 2015; Sato et al., 2013; Sofowora et al., 2013; Strege, 1999; Colombo Migliorero et al., 2020).

Combretaceae belongs to medicinal important plant family containing different 20 genera from which Combretum and Terminalia have been studied for isolation and characterization of phytochemicals to evaluate their different pharmacological activities (Saadullah et al., 2020). The genus Conocarpus has two species named as, C. lancifolius and C. erectus (Saleh et al., 2012). C. lancifolius belongs to an ornamental plant family and it grows usually in riverine and coastal regions of East Africa. It has been observed that this plant is also present in some regions of Pakistan (Baroon et al., 2012). On both surfaces of the leaves of C. lancifolius, trichomes and secretory ducts are present and the surface is protected by a thick cuticle layer exhibiting xerophytic characteristics. Their mature leaves are shiny and glossy in appearance. They have the leaves exhibiting xerophytic characteristics (Redha et al., 2011). It has been reported that Conocarpus genus contains different classes of secondary metabolites (Baroon et al., 2012). C. lancifolius grows in semi-arid and sandy soils environment. Secretory ducts are responsible for secretion of polyphenols, polysaccharides and epicuticular waxes.

The aim of this current study is to deal with isolation, elucidation and characterization of structure of new bioactive compound as well as determination of antioxidant, antiproliferative and anti-inflammatory activities of the novel compound. Reactive oxygen species (ROS) such as hydroxyl radical, superoxide anion radical, hypochlorous acid, hydrogen peroxide, ozone and superoxide radicals have an association with many disorders including carcinogenesis (Steer et al., 2002). ROS in the human body is produced as by-product of many metabolic reactions in the body (Navarro-Yepes et al., 2014). Many processes like activation of some factors that stimulate the transcription, phosphorylation of protein, and apoptosis are dependent and affected by suitable ROS generation and its level should be kept low inside the cells (Rajendran et al., 2014). Increased production of ROS induces harmful and unpredictable side effects on many important structural and functional components of the cell such as carbohydrates, nucleic acid, lipids and proteins (Taniyama and Griendling, 2003). Many studies in literature proved that oxidative stress is the leading cause of various disorders including metabolic and neurological disorders, cardiovascular diseases, atherosclerosis and cancer (Pizzino et al., 2017). Cancer is a worldwide health problem that may be due to exposure of carcinogenic agents such as toxins, virus, radiation and chemicals that cause uncontrolled growth of cells (Cohen et al., 2019). ROS are also responsible for induction of cancer by different mechanistic pathways such as proliferation, apoptosis and survival of cells, metabolism of energy, adhesion of cell–cell and motility of cells (Liou, G.Y. and P. Storz, 2010). Many other factors such as malnutrition, genetic variability, intake of alcohol and tobacco, lack of physical activities and some toxins are also major cause of immune suppression and development of cancer (Vineis et al., 2014). The number of cancer cases is steadily rising, driven by both rapid population growth and increased life expectancy. Projections indicate a 75 % surge in cancer cases by the year 2030 (Organization, W.H., 2016). Hence, it is crucial to explore innovative approaches for cancer treatments to alleviate patient distress and mitigate the escalating expenses associated with current costly therapies. While there is a wide array of medications targeting different cancer types, none are entirely foolproof or devoid of risks. A primary drawback of chemotherapy lies in its toxicity to healthy cells in the body, leading to significant side effects like fatigue, hair loss, anemia, and susceptibility to bruising and bleeding, among other issues (Prakash et al., 2013). Natural products and their derivatives exhibit significant promise in the creation of chemotherapeutic agents, showcasing extensive structural diversity and pharmacological and molecular attributes conducive to their advancement in this field (Pan et al., 2012). Cell lines derived from human tumors have traditionally played a crucial role in the development of novel cancer treatments. High-throughput cell-based profiling played a pivotal role across various discovery platforms but also in uncovering multiple agents later confirmed for therapeutic effectiveness. Substantial genomic diversity in human cancer and its impact on treatment response underscores the necessity to reevaluate the scope of cell line-based investigations for evaluating the efficacy of potential anticancer agents. As a result, more extensive arrays of cancer cell lines are now being utilized to uncover genomic factors influencing drug sensitivity. Recent literature has reinvigorated endeavors to employ cancer cell lines for evaluating the clinical viability of emerging investigational cancer drugs and identifying predictive biomarkers (Sharma et al., 2010). The aim of this study is to emphasize the untapped cytotoxic potential of C. lancifolius extract and isolated compound (lancifolian) against four human cancerous cell lines including MCF-7, A549, COR-L23 and MDA-MB-231.

Inflammation is the natural and immediate response of living organisms to ant kind of injury. This is defense mechanism of body to eliminate the chances of spread of infectious agent (Ofuegbe et al., 2014). There are different components of inflammation including leukocyte infiltration, granuloma formation and edema (Ofuegbe et al., 2014). The occurrence and uncontrolled progression of inflammation cause various inflammatory diseases leading to failure of body organs (Mehrzadi et al., 2021). For treatment of this adverse event, there is need of pharmacological interventions to improve the quality of healthy life (Archer et al., 2015).

Current therapeutic approaches for inflammatory disorders often involve synthetic drugs, which, despite their effectiveness, frequently come with a range of undesirable side effects and serious adverse reactions. These can encompass issues such as gastrointestinal disturbances, immunosuppression, and potential organ toxicity, making long-term use challenging for patients. The increasing recognition of these drawbacks has spurred a growing interest in herbal medicine as an alternative. Herbal remedies with anti-inflammatory properties are gaining prominence due to their perceived safety profile and the comparative absence of the adverse effects associated with many synthetic drugs. Exploring herbal alternatives becomes crucial for developing therapies that not only effectively address inflammatory conditions but also minimize the risk of debilitating side effects, offering a more holistic and potentially safer approach to long-term patient care. Numerous synthetic drugs once utilized for treating inflammatory disorders have fallen out of favor due to their associated potential side effects and serious adverse reactions, rendering them less desirable for human use. In recent decades, there has been a resurgence of interest in herbal drugs as viable alternatives for addressing various human ailments. Herbal remedies with anti-inflammatory activity have become particularly attractive due to their perceived safety profile compared to synthetic counterparts. In-depth examination of newly explored anti-inflammatory agents encompassing diverse classes of phytoconstituents. Herbal constituents undergo quite simple mechanisms that lead to fewer adverse effects. The potential interactions between herbal remedies and synthetic preparations, addressing associated adverse effects and summarizing clinical trials conducted on herbal substances proved fruitful results in anti-inflammatory activity (Beg, S., et al., 2011).

The compounds that showcase antioxidant properties slow down the oxidation process. Many medicinal plants reduce oxidative stress and ultimately shield the human from many diseases (Irshad et al., 2021). C. lancifolius extract has proven antioxidant activity as stated in the previously published paper (Saadullah et al., 2020). There are large numbers of anticancer agents from natural origins available in the markets (Olano et al., 2009). However, researchers are in a continuous search for characterization and isolation of anticancer agents from natural origins because of their high specificity towards cancer cell lines (Siddique et al., 2022). According to recent knowledge, no such report is published that investigated the anti-inflammatory effect of C. lancifolius extract to mitigate the edema of rat paw which is induced by carrageenan.

The main objectives of this study to isolate novel compounds from extract of C. lancifolius by multiple fractions that make this study more worthy. Spectral studies are used to identify the structure of novel compounds. The pharmacological activities of isolated compounds and extracts of C. lancifolius are investigated. To obtain the authentic information about the molecular aspects of this novel compound, molecular docking was performed This current project is designed by combining experimental and in silico computational analysis for evaluation of antioxidant, anti-inflammatory and cytotoxicity activity of methanol and dichloromethane extracts of C. lancifolius and lancifolian (novel isolated compound) obtained from C. lancifolius by extraction and purification for validation of our hypothesis. Antioxidant potential was evaluated by conducting in vitro test: DPPH (2,2-diphenyl-1-picrylhydrazyl) test, FRAP (ferric reducing antioxidant power) assay, ABTS (2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) assay and β-carotene bleaching assay while anti-inflammatory effect was observed via rat paw edema induced by carrageenan. Similarly, antiproliferative activity is evaluated against human cancer cell lines. This study will be helpful for researchers to attain their attraction on evaluation of pharmacological activities of natural products.

In previous studies, lancifolius shows cytotoxicity against MRC-5 cancer cell line. Isolated novel compound lancifotarene showed cytotoxicity towards cancer cell lines including murine lymphocytic leukemia (P-388), human colon cancer (Col-2), human breast cancer (MCF-7), while no cytotoxicity was observed towards human lung cancer (Lu-1), rat normal glioma cells (ASK) and human embryonic kidney cells (Saadullah at al., 2020).

The originality of this research lies in the isolation and purification of a novel compound named lancifolian from C. lancifolius. This compound is subjected to in vitro antioxidant assays, revealing its significant free radical scavenging ability. The study also highlights the anti-inflammatory effects demonstrated by both the C. lancifolius extract and lancifolian, determined through carrageenan-induced paw edema. Additionally, the novelty extends to the observed substantial cytotoxic effects against four different cancer cell lines (MCF-7, A549, COR-L23, and MDA-MB-231). The utilization of molecular docking techniques adds another layer of innovation, predicting the anti-inflammatory and cytotoxic properties by assessing the binding affinity of lancifolian with respective receptors. The validation of these molecular docking predictions further enhances the credibility and significance of the findings, contributing to the overall originality and novelty of the research.

2 Materials and methodology

2.5

2.5 Purification of lancifolian

Dichloromethanolic extract (12 gm) of C. lancifolius (CLD) was operated by column chromatography with aid of silica gel 60 of 40–63 μm (stationary phase) and mixture of chloroform and methanol used as mobile phase in the ratio as mentioned in Fig. 1 followed by alone use of methanol. The five fractions were obtained which were named as CLD-1 (1.10 gm), CLD-2 (4.27 gm), CLD-3(1.94 gm), CLD-4 (680 mg), CLD-5 (300 mg). The column chromatography was further performed to get more fractionations of 1.954 g of CLD-3 with silica gel 60 of 40–63 μm working as stationary phase and mobile phase is a mixture of ethyl acetate and hexane with ratio and sequence as shown in Fig. 1 By this operation, four more fractions were obtained named as CLD-3a + 3b (555 mg), CLD-3c (420 mg), CLD-3d (45 mg) and CLD-3e + 3f + 3 g (300 mg). The CLD-3e + 3f + 3 g (300 mg) fraction was again fractioned by column chromatography using silica gel 60 of 40–63 μm as stationary phase and mixture of chloroform and methanol was used as mobile phase. By this process, four more fractions were obtained which were named as CLD-3d-a (7 mg), CLD-3d-b (11 mg), CLD-3d-c (7 mg) and CLD-3d-d (2d mg). The 27 mg of CLD-3d-4 + 5 fraction was obtained and referred as novel compound (lancifolian). The schematic presentation of purification of dichloromethane (DCM) extract of C. lancifolius to obtain lancifolian is shown in Fig. 1.

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Fig. 1

Schematic presentation for purification of novel compound (Lancifolian) from C. lancifolius extract.

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3 Results

3.1

3.1 Structural elucidation

Lancifolian (7 mg) was obtained by from DCM fractions of C. lancifolius as coarse powder and appeared as colorless gummy solid having melting point 138–145 °C. Ultraviolet (UV) spectrum of lancifolian shows the absorption peaks at 222 and 230 nm (Fig. 2(a)) which depicts that conjugated system is present. IR spectrum of compound was represented in Fig. 2b. IR spectrum of lancifolian shows absorption peak at 3419.6 cm⁻¹ for –OH. The peak at 2928.3 cm⁻¹ is for C-H stretching (Sp² hybridized). IR spectrum shows absorption at 2125.4 cm⁻¹ shows carbonyl group present in the compound. C = C indicates by presence of absorption peak at 1513.1 cm⁻¹. Absorption peaks at 1513.1 cm⁻¹ shows that lactone moiety is present in compound. The proton NMR (¹H NMR) spectra of isolated compound shows the signals at δ 5.78 (1H, singlet) and δ 4.71 (1H, dt having J = 4.2, 2.4 Hz) for alkene and alcoholic proton. The peak at δ 4.58 (1H, d having J = 6.3 Hz) shows the presence of lactone moiety in compound (Fig. 2(c)). ¹³C NMR spectrum (Fig. 2(d)) shows broad band and depth. The compound in ¹³C NMR spectra shows the presence of fourteen carbon signals: six for hydroxyl group, seven for methane and one for methylene (Fig. 2(d)). ¹³C NMR spectra also show the five quaternary carbon signals. In the downfield region, singlet signals at δ 189.9 indicate the presence of carbonyl carbon in compound. The signal in ¹³C NMR spectrum at δ167.9 indicates lactone carbons exist in compound. Alkene carbons also predicted in the structure due to presence of signals at δ 114.9, 115.9 and 130.7 cm⁻¹. The molecular formula C₁₄H₁₆O₉ of compound was elucidated using high-resolution electron ionization mass spectrometry (HR-EI-MS) showing peak for isolated and purified compound at 328 (m/z). Fast atomic bombardment (FAB) and electron ionization (EI) spectrum for confirmation of structure and fragmentation pattern is shown in Fig. 2e and 2f. The connectivity information of proton in two-dimensional perspective, correlation spectroscopy (COSY) and HQSC is shown in Fig. 2g and h. All mentioned physical and spectral data (Fig. 2(a–h)) confirm the identification of compound, 1,3,4,5,6,8-hexahydroxy-3,4,5,5a-tetrahydro-1H-benzo[g]isochromene-5-carboxylic acid which is named as lancifolian. Table 1 presents the summarized physical and spectral data of isolated and purified compound (lancifolian). Fig. 3 presents the structure and fragmentation pattern of lancifolian. The multiplicity of carbon is shown by DEPT tool in Table 2.

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Fig. 2

Spectrum for elucidation of lancifolian structure. 2(a): UV spectrum of lancifolian, 2(b): IR spectrum of lancifolian, 2(c): ¹H NMR spectrum of lancifolian, 2(d): ¹³C NMR broad band (BB) spectrum of lancifolian, 2(e): FAB spectrum of lancifolian, 2(f): EI spectrum of lancifolian, 2(g): COSY spectrum of lancifolian, 2(h): HQSC spectrum of lancifolian.

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Table 1 Physical and spectral data of isolated compound (Lancifolian).

Characteristics	Results
Color	Colorless gummy solid
Quantity	7 mg
Melting point	Ranging from 138 to 145 °C
[α]D25	62.0° (0.12).
Ultaviolet λmax;	Shows the maximum absorption at 230 and 222 nm.
Infrared (KBr)	Shows the peaks for different functional groups: 3419.6, 2928, 2125.4, 1652 and 1531 cm−1.
1H NMR (600 MHz)	Proton resonate at chemical shift δ 4.56 (1H, d having J = 7.5, H-1́), δ 5.79 (1H, s for 5 – OH), δ 6.72(1H, d having J = 1.4 Hz for H-4), δ 6.85(1H, d having J = 1.6 Hz, H-2), δ 6.87(1H, d having J = 1.3 Hz, H-6), δ 7.35 (1H,dd, J = 6.3, 2.7 Hz, H-4), δ 7.4 (1H, J = 6.3 Hz, H-5)
13C NMR (150 MHz)	Carbon shows chemical shift δ at 61.7, δ 70.3 (C-4″), δ 73.8 (C-2″), δ 76.8 (C-3″), δ 77.4(C-5″), δ 95.8 (C-8), δ 100.5(C-6), δ 100.7, δ 103.7 (C-3), δ 106.3, δ 114.8 (C-2″), δ 115.5 (C-5′), δ 130.3, δ 122.6, δ 166.9 (C-7), δ 167.8(C-2) and at δ 168.1.
EI-MS	(relative intensities measure) m/z 328 (72), 252 (1 8 5), 224 (20), 212 (15), 196 (20), 146 (19), 73 (14)
HR-EI-MS	(m/z): molecular formula (C14H16O9), molecular weight (MW) 328.08

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Fig. 3

Illustrates the structure and fragmentation pattern of lancifolian, by providing a visual representation of its molecular configuration and the resulting fragments.

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Fig. 4

The surface view, pose view, 2D and 3D interactions study of co-crystal ligands (2AZ5 (4(a) and 4ASD 4(b)) lancifolian. The redocking process of lancifolian with crystallography (red: result of co-crystal; green: result of redocking) showing the superimposing position of the ligand (4(c).

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Table 2 Distortionless Enhancement by Polarization Transfer of carbon of lancifolian.

C-No.	Multiplicity DEPT	13C NMR	1H NMR	J –value
C-1	–CH	95.4	5.26	(J = 1.4 Hz)
C-3	–CH	101.3	4.69	(J = 1.6 Hz)
C-4	–CH	70.4	4.31	(J = 2.4 Hz)
C-5	-C	79.6
C-6	-C	181.8
C-7	–CH	103.0	3.64	(J = 1.4 Hz)
C-8	–CH	64.8	3.94	(J = 2.1 Hz)
C-9	–CH2	41.7	2.27, 2.02	(J = 1.3 Hz)
C-10	–CH	126.9	2.24	(J = 2.2 Hz)
C-11	-C	132.8
C-12	-C	135.9
C-13	-C	144.8
C-14	–CH	45.5	3.23	(J = 2.3 Hz)

4 Discussion

C. lancifolius is medicinally important plant that belongs to Combretaceae family (Ali et al., 2013). Different studies have proved that conocarpus genus possesses potential therapeutic activities such as antiprotozoal, anti-leishmanial, antibacterial and anticancer. This study focuses on the antioxidant, anti-inflammatory and antiproliferative characteristics of C. lancifolius. A novel compound, lancifolian was isolated from DCM extraction of C. lancifolius and proved as antioxidant, anti-inflammatory and anticancer agent. Purification of novel compound was done by multiple fractionations using column chromatography where stationary phase of silica gel 60 (63–200 μm) and mixture of methanol and chloroform was used as mobile phase.

Moreover, the structural elucidation and spectral analysis lancifolian was also performed. The UV and IR spectra show conjugation and existence of aromaticity in compound. IR spetra also shows the presence of hydroxyl group, carbonyl group and lactone moiety. ¹H NMR spectrum shows the signals to depict the presence of protons of hydroxyl and alkene group. ¹³C NMR shows the presence of fourteen signals for carbon in which six for hydroxyl group, one for methylene while seven for methane group. ¹³C NMR also depicts five signals for quaternary carbon. Hence, structure elucidation of lancifolian was performed by HR-EI-MS and determined the molecular formula of novel compound C₁₄H₁₆O₉ named as lancifolian.

Free radicals are species having unpaired electrons and produced in the body either exogenously (ionizing radiation, organic solvents, pesticides and air pollution) or endogenously (as a by-product of metabolism). Increased level of ROS has potential to induce tissue injury and death of cell (Chanda et al., 2009). Damage induced by ROS leads to development of various chronic diseases including neural disorders, cardiovascular disease, cognitive impairments, atherosclerosis, cancer and ulcerative colitis (Hossain et al., 2015). Antioxidant activity of phytoconstituents are due to redox potential which neutralize the free radicals species, decompose peroxides and quench singlet and triplet oxygen species (Pietta et al., 1998). Antioxidant potential of molecule is proportional to hydroxyl groups present in a molecule (Norma Francenia et al., 2019). Lancifolian has antioxidant potential due to the existence of hydroxyl group in its structure. It was evaluated by in vitro antioxidant tests (Hossain et al., 2016).

Inflammation-induced by carrageenan is mainly due to inflammatory mediators including prostaglandins, leukotriene, platelet activating factors (PAF) and others inflammatory mediators. The inflammation induced by carrageenan can be described as biphasic phenomena. In the first pahse (0 to 1 h), histamine, kinin and hydroxyl tryptamine (5-HT) releases. In the second phase (3 to 5 h) is endorsed to release of bradykinin and prostaglandin (Mehrzadi et al., 2021). 200 mg extract of C. lancifolius exhibited more anti-inflammatory effect than 100 mf extract of C. lancifolius. The results show the direct correlation of dose strength in reduction of inflammation induced by carrageenan. The extracts and isolated compound exhibited excellent results in reduction of inflammation by significantly alter the prostaglandins mediated response which was disturbed by carrageenan (Fakhar-e-Alam et al., 2023).

In vitro cytotoxic activity of lancifolian, DCM and methanolic extract of C. lancifolius was estimated. Cytotoxic activity of C. lancifolius was investigated against four specific cancer cell lines: MDA-MB-231 and MCF-7 while taxol is used as positive control and A549, COR-L23 where vinblastine sulphate is used as positive control. Lancifolian shows the best anticancer activity (IC50 = 78.4 ± 2.5) in comparison to DCM extract of C. lancifolius (IC50 = 151.4 ± 3.2) while methanolic extract does not exhibit significant anticancer activity for MCF-7 cell lines where taxol is used as positive control (IC50 = 0.06 ± 0.003). Lancifolian and DCM extract does not exhibit anticancer activity against A549 cell lines while methanolic extract exhibit anticancer activity (IC50 = 78.1 ± 2.4) where vinblastine sulfate used as positive s 82.3 ± 2.6 and 164.3 ± 3.2 respectively while methanolic extract does not show anticancer activity where taxol is considered as positive control (IC50 = 1.4 ± 0.02). For COR-L23 cell lines, only lancifolian shows anticancer activity (IC50 = 112.5 ± 2.3) while vinblastine sulphate is used as (IC50 = 44.2 ± 0.04) positive control. Almost, similar results for antiproliferative activities of Coronopus didymus leaf extract were found in literature (Muzammil et al., 2022a).

We also confirmed the binding affinity of lancifolian with receptor and determined the binding energy of lancifolian and receptor complex. In literature, site-specific docking was also performed on different reported inhibitors in T. divaricata (Muzammil et al., 2022b). The results indicated that ligand showed the best interaction with 2AZ5 and 4ASD amino acid residues of receptors. Revalidation of molecular docking was also performed (Malik et al., 2022). The results of revalidation confirmed that ligand and receptor complex have lower RMDS value indicates the position of redocking ligand is near to crystallographic ligand location.

Comparing these results with existing research, this study offers comprehensive insights into the therapeutic potential of C. lancifolius, particularly through the novel compound lancifolian. The robust experimental design detailed structural elucidation, and in-depth analysis of antioxidant, anti-inflammatory, and anticancer activities contribute to the study's strength. Future research could explore the synergistic effects of the plant extracts, conduct clinical trials, and investigate the pharmacokinetics of lancifolian for a more translational perspective.

5 Conclusion and future recommendation

This research has concluded the multicomponents from extract of C. lancifolius. The isolation and purification of novel compound (lancifolian) from C. lancifolius was done using chromatography and spectroscopic techniques. The antioxidant and cytotoxic activity of C. lancifolius extracts and isolated compound was confirmed through in vitro analysis and anti-inflammatory effect was also evaluated by conducting experiment on animal model. We confirmed the cytotoxic and anti-inflammatory activity of lancifolian by using computational tool and molecular docking. The results of our study confirmed that extracts of selected plant and isolated compound from C. lancifolius have potential to inhibit the oxidative stress, reduce the inflammation and kill the cytotoxic cells when compared with standards. Due to cytotoxic potential of lancifolian, it inhibits metastasis by causing death of cancerous cells. Lancifolian exhibited antioxidant and anti-inflammatory activity ultimately preventing the development of many diseases. Besides the significant results from in vitro analysis, molecular docking was performed to confirm the binding affinity of lancifolian with anticancer and anti-inflammatory receptors. Based on results computed by different experimental models and in silico studies, lancifolian could be recommended as best cytotoxic, anti-inflammatory and antioxidant agent. The exploration of bioactive compounds from C. lancifolius within the Combretaceae family holds immense potential for future advancements in medicine. The isolation and purification of the novel compound, lancifolian, derived from this plant, opens doors to extensive research opportunities. To further unravel its therapeutic possibilities, future investigations could focus on elucidating the specific molecular mechanisms underlying notable antioxidant effects. Clinical trials are warranted to validate the observed anti-inflammatory properties and explore the safety and efficacy of C. lancifolius. Additionally, the significant cytotoxic effects against various cancer cell lines necessitate in-depth in vivo studies for a comprehensive understanding of their anticancer potential. Optimizing extraction techniques and exploring potential synergies with existing therapeutic agents could enhance the bioavailability and efficacy of these compounds. Further validation of molecular docking predictions and the development of lancifolian derivatives could contribute to refining its application in the treatment of various diseases. Continuous collaboration with local communities for ethnobotanical insights and comprehensive long-term safety studies will be essential for harnessing the full medicinal potential of C. lancifolius and its bioactive compounds.

While the study on C. lancifolius and its bioactive compound lancifolian presents promising findings, several challenges, and limitations warrant consideration. Firstly, the in vitro assays and animal model experiments, while indicative of potential therapeutic effects, may not precisely replicate the complexities of human physiology. Additionally, though molecular docking enhances predictions, it relies on computational models and assumptions. The isolation and purification process of lancifolian, while utilizing chromatography and spectroscopic techniques, could benefit from further optimization for increased yield and purity. Furthermore, the study primarily focuses on individual components, and understanding potential synergies or interactions within the plant extracts could provide a more comprehensive picture. Future research should aim to conduct rigorous clinical trials to validate the observed effects in humans and assess long-term safety. Moreover, exploring the pharmacokinetics and pharmacodynamics of lancifolian would contribute to a more thorough understanding of its therapeutic potential. Collaborative efforts between researchers in the fields of natural products, pharmacology, and clinical medicine can enhance the translational impact of this study, facilitating the development of lancifolian as a potential therapeutic agent for various diseases.

CRediT author statement

Malik Saadullah: Conceptualization, Data curation, Writing – original draft. M. Fakhar-e-Alam: Conceptualization, Data curation, Writing – original draft. M. Atif: Formal analysis. Muhammad Asif: Writing – review & editing. Kanwal Irshad: Conceptualization, Data curation, Writing – original draft. Zulfiqar Ali: Formal analysis.