Anticancer potential of mangrove derived metabolites: cytotoxicity and phytochemical based studies.

Background
Mangrove forests represent a vast biological diversity of plants, microorganisms, and animals. They are found in the intertidal zones and serve as a bridging ecosystem between freshwater and marine systems. As mangroves grow in extreme conditions, they tolerate environmental stressors such as salinity, temperature gradients, tidal fluctuations, strong UV exposure and anoxic soil conditions. These harsh conditions trigger unique metabolic pathways that enhance the synthesis of diverse bioactive compounds that protect them from these destructive elements. The metabolites act as defense molecules against pathogens and oxidative stress [1]. Mangrove plants are spread in 123 tropical and subtropical nations worldwide, with 84 species belonging to 24 genera and 16 families. They are broadly categorized into 2 groups based on their habitats in nature, namely: true mangroves (specifically grow in intertidal zones) and semi-mangroves (capable of growing in either littoral or terrestrial habitats) [2]. Among them, 70 species are of true mangroves belonging to 16 genera and 11 families, whereas 14 species are of semi-mangroves belonging to 8 genera and 5 families [3]. The most common and widely distributed species of mangrove plants belong to the Rhizophoraceae family, comprising of 24 species across 4 genera, namely, Bruguiera (containing 7 species), Ceriops (containing 5 species), Kandelia (containing 2 species) and Rhizophora (containing 10 species) [4]. The genus Bruguiera has a metabolic pattern that has been marked by the presence of diterpenes and triterpenes and characterized by the presence of disulphides and polysulphides [4, 5]. The plants belonging to the genus Ceriops are valued for their rich tannin and pentacyclic triterpenoid content [6]. The Rhizophora genus has a variety of diterpenoids, triterpenoids, and steroids. A few diterpenoids, such as beyeranes, were identified from Rhizophora mucronata and have proven to be unique to this species [7]. For centuries, mangrove plant extracts have been used as traditional medicine for healing health-related disorders by tribal populations. As of now, various mangrove plants have been investigated and identified as potential sources of novel natural compounds for use in medicine. In fact, the majority of identified compounds are unique and demonstrated interesting biological activities such as anti-inflammatory, gastroprotective, cytotoxic, antioxidant, antibacterial, antifungal, antiviral, enzyme activation and inhibition, immunosuppressive, and antifeedant effects [6, 8]. The secondary metabolites that are accumulated in high concentrations under stress conditions by mangrove plants belong majorly to phenolics, alkaloids, and terpenoids [9]. Natural products derived from the plants, such as flavonoids, terpenes and alkaloids have gained attention for their cytotoxic and cancer-preventive properties [10–12].
Cancer remains a major global health threat, causing substantial mortality and imposing substantial economic and health-related costs worldwide [13]. In 2022, there were almost 10 million cancer-related deaths and close to 20 million new cancer diagnoses. With an estimated 2.5 million new cases (12.4% of all cancers), lung cancer continued to be the most often diagnosed condition. It was also the largest cause of cancer-related mortality, accounting for an estimated 1.8 million deaths (18.7%). Oral cancer is the sixteenth most common cancer globally, with an estimated 389,485 new cases and 188,230 deaths per year [14]. The highest incidence rates are observed in South and Southeast Asia, particularly in India, Sri Lanka, Bangladesh, and Pakistan, where oral cancer accounts for up to 40% of all cancers. In India, it represents nearly 30% of all cancer cases, making it the most frequently diagnosed cancer among men [15].
Chemotherapy remains the primary therapeutic option for cancer patients, particularly those in the last stages of the disease. Nonetheless, the development of drug resistance and significant side effects limit the use of chemotherapy for cancer treatment. The initial appearance of the chemotherapeutic issues is primarily due to drug inactivation and metabolic biotransformation by several enzymes, including cytochromes P450. Identifying the molecular mechanisms that cause the above mentioned issues is still an important subject of study that can assist in uncovering novel pharmacological drug targets and developing new drug leads, particularly from natural products, to enhance patients' treatment results [16]. Studies have discovered that secondary metabolites originating from plants can specifically induce cancer cells to undergo apoptosis, reduce metastasis and prevent proliferation, making them a promising and reliable candidate for the development of anticancer drugs [17]. Compounds such as Tagalon C, Tagalon D, Tagalene I, Tagalene K, and Tagalsin C have been extracted from C. tagal and have proven to exhibit cytotoxic effects against MDA-MB-453, MDA-MB-231, SK-BR-3, MT-1, SW480, HeLa, PANC-1, HCT-8, Bel-7402, BGC-823, A549, and A2780 cell lines with IC50 values ranging from 3.72 to 8.97 μM [18]. In a previous study, certain compounds extracted from R. mucronata, such as 4-O-caffeoyl quinic acid, were noted as the most active compound among those tested against ovarian (SKOV3) and colorectal (HT29) cancer cell lines with IC50 values of ≤ 20 µg/mL indicating potent effects. Other compounds that were tested include 5-O-caffeoyl quinic acid, which demonstrated strong inhibitory effects against ovarian (A2780) cancer cell lines, epi-catechin showed significant anticancer properties against various cell lines, including breast (T47D), colorectal (HT29), and ovarian (A2780, SKOV3) [19]. Even though extracts from mangroves have been acknowledged for their cytotoxic potential, the specific compounds responsible for these bioactivities have not been entirely characterized [20]. Thus, our research aims to address this gap by evaluating the cytotoxic potential of the R. mucronata, R. apiculata, B. cylindrica, B. gymnorhiza, C. tagal and K. candel leaf extracts against AW 13516, A549 and HeLa cancer cell lines using the MTT assay and monitor the associated anti-migratory properties using the scratch wound healing assay. Liquid chromatography-high resolution mass spectrometry (Orbitrap-LC-HRMS) was used to identify specific compounds present in the mangrove leaf extracts that may be responsible for the anticancer activity.

Materials and methods

Sample collection
Leaves of 6 different mangrove species belonging to the Rhizophoraceae family were collected from the naturally growing stands of the mangrove vegetation in Chorao Island, Goa, which is situated on the West Coast of India at a geographical latitude of 15.54°N and longitude of 73.88°E. The collected mangrove species included K. candel, C. tagal, B. gymnorhiza, B. cylindrica, R. mucronata, and R. apiculata. The mangrove species were identified based on morphological keys and verified by Dr. Manoj M. Lekhak (Department of Botany, Shivaji University, Kolhapur). The herbarium specimens bearing voucher numbers AAS-190–92, AAS-200–02, AAS-140–42, AAS-130–32, AAS-150–52 and AAS-160–62 corresponding to K. candel, C. tagal, B. gymnorhiza, B. cylindrica, R. mucronata, and R. apiculata, respectively were deposited at Botanical Survey of India, Western Regional Centre, Pune, India [21, 22]. Samples of mangroves were obtained with prior permission from the office of Chief Conservator of Forest, Goa Forest Department, India. Moreover, none of the samples belong to endangered or protected species.

Preparation of leaf extracts
The leaves were thoroughly and carefully washed under running tap water and then dried for 21 days in a shaded and ventilated location at a room temperature of 27 °C. It was observed that different leaf samples were drying at different rates because of the difference in their morphologies; hence, after 21 days, the samples were closely monitored every 24 h for a reduction in their weight. The dry leaves were then crushed into a fine powder with the use of an electric grinder and stored in airtight bottles in a cool and dark place. 30 g of each sample was extracted with 300 mL (a solvent-to-plant material ratio of 1:10 has been reported in previous studies as the most suitable proportion for Soxhlet extraction, yielding optimal extraction efficiency and reproducible results) of methanol using the Soxhlet apparatus (Sunbim, India) for 24 hours [23]. Soxhlet extraction ensues continuously until secondary metabolites are fully extracted without replenishing the solvent, signified by the absence of pigmentation in methanol exiting the extraction chamber [24]. Methanol facilitates the efficient recovery of diverse bioactive compounds, and due to high polar nature, it serves as a potent solvent for extraction of the secondary metabolites including terpenoids, phenolics, and flavonoids [25, 26]. The different extracts obtained were then passed through Grade 1 Whatman filter paper and evaporated in a rotary vacuum evaporator (Roteva, Medica Instruments, India) at a temperature lower than 40 °C. The crude methanolic extracts were dried and stored at −20 °C until further analysis.

Maintenance of cell lines
The animal cell culture laboratory at BITS-Pilani K.K Birla Goa Campus, India, provided AW 13516 (derived from poorly differentiated squamous cell carcinomas (SCC) of the oral cavity), A549 (hypotriploid alveolar basal epithelial cells), HeLa (cervical cancer cells), and HaCaT (human keratinocyte cell line) cells. A laminar airflow cabinet was used to maintain the sterility of the process. The Dulbecco’s Modified Eagle medium (DMEM) (Himedia) was used to sustain the cell culture containing 1% antibiotic and antimycotic solution (10,000 U penicillin, 10 mg streptomycin and 25 µg amphotericin B per mL in 0.9% normal saline) (Himedia) and 1% gentamicin solution (Himedia) [27]. 10% fetal bovine serum (Heat Inactivated) (Himedia) was used as a supplement. The cells were incubated at a temperature of 37◦C for 24 h in a humidified condition of 5% CO2 incubator (Thermo Scientific) to allow the cells to grow. The cells were monitored under an inverted microscope (EVOS XL Core Imaging System- Thermo Fisher Scientific, Carlsbad, CA, USA) for confluency and checked for possible contamination before initiating further experiments. The cells were incubated and passaged using trypsin–EDTA (0.25%), then centrifuged at 2000 rpm for 3 min. The pellet was resuspended in DMEM media (1 mL) for further analysis [28].

Evaluation of cytotoxicity of leaf extracts by MTT assay
For the evaluation of cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay was employed. Briefly, 1 × 104 cells per 100µL of DMEM were cultured in 96 well plates. The plates were incubated for 24 h at 37 °C and 5% CO2 so that a uniform monolayer sheet is formed. After the cancer cells form a confluent layer, the growth medium was aspirated from the wells of the 96 well plate. Initially, dimethyl sulfoxide (0.2%) (Himedia) was used to dissolve the methanolic leaf extracts which was further serially diluted by using DMEM to make the final concentrations. 100µL of the extract (done in triplicates) at each concentration was dispensed in the 96 well plate and only DMEM was added to the control cells. Again, the plate was incubated for 24 h at 37 °C and 5% CO2. Phosphate-buffered saline (Himedia) was used to dissolve the MTT powder by vortexing to a concentration of 5 mg/mL. It was further diluted to a concentration of 0.5 mg/mL with the help of DMEM. After the completion of the incubation period, 100µL of the MTT solution was added to the wells after aspirating the previously added extract solution. It was again kept in the incubator for 4 h at 37 °C and 5% CO2. Through mitochondrial reduction, a purple formazon is created from yellow coloured MTT. Formazon was resuspended in 100 µL of DMSO and the plate was shaken for 5 min. The wavelength in the microplate reading spectrophotometer was set to 570 nm and the optical density was recorded. Experiments were conducted in triplicates for the studied cell lines and % growth inhibition was evaluated. Percent growth inhibition of cells exposed to methanolic extract treatments was calculated as follows:  and there IC50were calculated via GraphPad Software, Inc [29].

Investigation of cell migration by in vitro wound healing scratch assay
The scratch wound healing assay is one of the effective and standard methods to analyse the cell migration of cancer cells upon treatment. This method was employed to calculate the rate of migration of the A549 cells when treated with the prepared methanolic leaf extracts in comparison with the untreated cells. The measurements were taken for 0 h, 6 h, 12 and 24 h treated cells in order to minimize the contribution of cell proliferation to fill the gap.
For this purpose, A549 cancer cells were seeded in a 6 well plate (2 × 105 cells/well) so that a 80% confluent monolayer was obtained after an incubation period of 24 hours in a humidified condition of 5% CO2 incubator. The cell monolayer was scraped in a straight line by using a sharp object such as a 200 µL pipette tip. It is of utmost importance to create scratches of similar size in order to minimize variations due to width differences. The cells were washed with 1 X PBS (pH-7.4) solution in order to remove the suspended floating cells so that the edges of the scratched surfaces are clean. After scratching, the cells were reincubated with the doses of methanolic leaf extracts of B. gymnorhiza (400.226 µg/mL), B. cylindrica (323.965 µg/mL), C. tagal (329.764 µg/mL), K. candel (397.792 µg/mL), R. apiculata (566.283 µg/mL) and R. mucronata (399.05 µg/mL) and the negative control contained cells with just DMEM. The area of the gap of all tested and control cells were measured at 0 h, just after the scratch followed by gap area measurements after 6 h, 12 h and 24 h of treatments. The scratch closure was observed under inverted microscope (EVOS XL Core Imaging System- Thermo Fisher Scientific, Carlsbad, CA, USA) at 10X magnification and the images were captured. Further, the migration % was calculated as [28]:-

Selectivity index
The response of HaCaT (Non-Cancer) cells and cancer cells for each extract were compared using the selectivity index (SI). The selectivity index (SI), according to the literature, may be defined as the ratio of the IC50 value of the plant extract in a non-cancer cell line (HaCaT) to its IC50 value in each cancer cell line. It determines the selectivity of the plant extract towards the cancer cells. An SI value greater than 1 indicates that the plant extract has in vitro selective activity against cancer in relation to normal (non-cancer) cells, whereas a value less than 1 indicates that the plant extract is not selective for cancer cells relative to normal cells [30].

Phytochemical screening through LC-HRMS Orbitrap analysis
The secondary metabolites from the samples were qualitatively analyzed using a high-resolution liquid chromatograph mass spectrometer (LC-HRMS Orbitrap). The instrument used was Q-Exactive Plus Biopharma with Hypersil Gold (100 × 2.1 mm, 3 µm) column (Thermo Scientific, USA). The two mobile phases, Solvent A: 0.1% formic acid in milli-Q water and methanol as solvent B, were used in a gradient condition of 300 µL/min flow rate. Linear gradient elution parameters were set as follows: initially from 0 to 2 min 5% B, 2–25 min 95% B, and 26–30 min 5% B. Prior to analysis, sample was prepared by following the protocol described [31] with minor modifications. Parent stock was prepared by dissolving 10 mg of sample in 1 mL of methanol and heated in water bath (50℃) to ensure its complete dissolution and then centrifuged (10,000 rpm, 8 min). Final working stock of 1 mg/mL was prepared in methanol and filtered through 0.22 μm nylon membrane filter (GE Healthcare, Chicago, IL, USA) before injection. The injection volume was 5 µL. Complete scan data was acquired in positive and negative modes at a resolution of 70,000 and a scanning range from m/z 100 to 1500. For data acquisition, Thermo Scientific Xcalibur (Version 4.2.28.14), and for data processing, Compound Discoverer 2.1 SP1 (Thermo Fisher Scientific, USA) software was used, which is a data-processing application that qualitatively processes the data based on accurate mass and mass spectral library searches for the identification of small molecules. The mass tolerance was set to 5 ppm. For the identification of the metabolites data was processed with the help of compound database such as mzCloud and ChemSpider.

Statistical analysis
All experiments were performed in triplicates and the findings were shown as mean ± standard deviation (n = 3). The comparative cytotoxicity profiles of the methanolic extracts from six mangrove species against A549, AW 13516, and HeLa cell lines were statistically analysed using a one-way ANOVA test followed by Turkey’s multiple comparison analysis to assess differences among the extracts. Significance levels were indicated as ****p < 0.0001; ***p < 0.001; **p < 0.01; *p ≤ 0.05; and ns (non-significant) p > 0.05. Similar statistical analysis was also performed in scratch wound healing assay in which the experimental data of the leaf extracts and untreated cells were compared.

Results

Evaluation of the cytotoxic potential of the mangrove extracts against AW 13516, A549, HeLa and normal human cell lines
The anticancer activities of the methanolic leaf extracts of R. mucronata, R. apiculata, B. gymnorhiza, B. cylindrica, C. tagal and K. candel were tested and compared against AW 13516, A549 and HeLa cancer cell lines by MTT assay. Cell viability analysis using the MTT assay demonstrated a dose-dependent suppression of cellular viability, wherein treated cells exhibited a progressive increase in % inhibition in direct correlation with increasing concentrations of the methanolic plant extract (Fig. 1). The effectiveness of an extract is inversely correlated with its IC50 value (half-maximal inhibitory concentration), such that, a lower IC50 value signifies the capacity to exert cytotoxic effects at decreased concentrations. IC50 (µg/mL) values of various methanolic extracts of mangroves against cell lines is given in Table 1. Our study revealed that the minimum half-maximal inhibitory concentration (IC50) value for the AW 13516 cell line was observed at a concentration of 287.062 ± 1.127 µg/mL of B. cylindrica at a 24-h duration. Conversely, the maximum IC50 value was recorded at 475.649 ± 2.549 µg/mL upon exposure to the methanolic extract of B. gymnorhiza. Moreover, we observed IC50 values of 360.921 ± 1.207 µg/mL, 408.354 ± 12.298 µg/mL, 411.447 ± 3.459 µg/mL, and 465.740 ± 3.634 µg/mL for C. tagal, R. apiculata, K. candel, and R. mucronata, respectively. Treatment of the oral cancer cells with methanolic leaf extracts at concentrations equivalent to the calculated IC50 values is depicted in Fig. 2C-H, indicating an inhibition of viable cell count. Morphological examination revealed a significant alteration in cell morphology post-treatment. The cells became elongated, contrasting with the typical epithelial cell morphology of untreated cells (Fig. 2A) [32]. Notably, a stressed-cell morphology similar to that observed with our study was replicable with the application of 8.20 µg/mL (IC50) doxorubicin as a positive control (Fig. 2B).
In A549 cell lines, the minimum half-maximal inhibitory concentration was identified as 323.965 ± 1.417 µg/mL after 24 hours exposure to the methanolic extract of B. cylindrica. In contrast, the highest IC50 value was exhibited by R. apiculata at 566.283 ± 3.250 µg/mL. Additionally, the IC50 values for other investigated species were determined as follows: 329.764 ± 0.653 µg/mL for C. tagal, 397.792 ± 0.546 µg/mL for K. candel, 399.05 ± 1.255 µg/mL for R. mucronata, and 400.226 ± 1.488 µg/mL for B. gymnorhiza. Microscopic observations of lung cancer cells subjected to methanolic leaf extracts at IC50 concentrations revealed a marked reduction in viable cell count (Fig. 3C-H). Treatment resulted in significant shrinkage and rounding (Fig. 3B), paralleling the morphological characteristics evident in cells exposed to doxorubicin (IC50 = 42.65 μg/mL), thereby validating the efficacy of the plant extracts. In HeLa cell lines, the minimal IC50 was observed at 25.942 ± 2.048 µg/mL of B. cylindrica after 24 h, while K. candel exhibited the highest IC50 value at 177.48 ± 12.594 µg/mL. Significantly, IC50 values of other tested extracts were found to be 73.147 ± 1.092 µg/mL for B. gymnorhiza, 79.354 ± 1.826 µg/mL for R. mucronata, 91.426 ± 2.660 µg/mL for C. tagal, and 115.55 ± 19.611 µg/mL for R. apiculata. The IC50 value observed for HeLa cell line after treatment with doxorubicin was 3 ± 1.945 μg/mL. In HaCaT cell lines, a minimum IC50 was observed at a concentration of 1556.636 ± 12.613 µg/mL of C. tagal at 24 h, while B. gymnorhiza exhibited the maximum IC50 value at a concentration of 2158.568 ± 17.812 µg/mL. The IC50 values of B. cylindrica, K. candel, R. mucronata, and R. apiculata were recorded as 1609.308 ± 12.135 µg/mL, 1865.130 ± 15.849 µg/mL, 1987.675 ± 17.079 µg/mL, and 2032.684 ± 18.910 µg/mL, respectively. Interestingly, in accordance with the Geran and National Cancer Institute (NCI) protocols none of the plant extracts exhibited any cytotoxic effect against normal cell lines [16]. These IC50 values signify the promising candidature of the methanolic leaf extracts in the development of a novel chemotherapeutic drug for the treatment of various human cancers as they have resulted in mitigating the cytotoxic impacts on normal human cells. The criteria used to categorize the cytotoxicity of plant crude extracts against cancer cell lines, based on U.S. National Cancer Institute (NCI) and Geran protocol was as follows: IC50 ≤ 20 µg/mL = highly cytotoxic, IC50 ranged between 21 and 200 µg/mL = moderately cytotoxic, IC50 ranged between 201 and 500 µg/mL = weakly cytotoxic and IC50 > 501 µg/mL = no cytotoxicity [16]. The comparative statistical analysis using one-way ANOVA indicated that there were highly significant interspecies cytotoxic activity differences (****p < 0.0001), while on the other hand only a few comparisons didn’t show any significant difference (ns) (Fig. 4).

Selective cytotoxicity of the mangrove leaf extracts against different cancer cell lines
Data from the selectivity index (SI) further validate the anticancer potential of the extracts against cancer cells, particularly B. gymnorhiza and R. mucronata, which demonstrated enhanced selectivity towards A549 cells with an SI value of 5.39 and 4.98, respectively (Table 2). B. cylindrica and R. apiculata exhibited selective cytotoxicity towards the AW 13516 cell line with an SI value of 5.60 and 4.97, respectively. Higher SI values suggest a more significant therapeutic window, where cytotoxicity is more pronounced in cancer cells while sparing normal cells [33]. Bioactive compounds exhibiting selectivity indices (SI) greater than 2 are frequently regarded as displaying substantial potential for targeted anticancer therapeutic applications, rendering B. cylindrica as a candidate worthy of further investigation due to its exceptional selectivity profile [34]. Notably, its pronounced selectivity against cervical cancer cells, evidenced by a high SI value of 62.03 (followed by B. gymnorhiza (29.51) and R. mucronata (25.04)), underscores its potential for selective therapeutic cytotoxicity in cervical cancer. Moreover, its concurrent efficacy against oral cancer cells positions it as a prime candidate for further probe in targeted therapies.

Antimigratory effects of the mangrove extracts on lung cancer cell line
In in vitro scratch wound healing assay, image analysis done at 0, 6, 12, and 24 hours post-scratch revealed a gradual closure of the wound area in the control group (Fig. 5 and Supplementary Figure 1). While the migration % of the treatment groups were significantly negligible by the end of 24 hours, the migration % of the control cells reached up to 86.54 ± 0.84% as shown in Table 3. A549 cells treated with the R. apiculata extract showed a significantly lower wound closure rate (- 45.58 ± 1.59%), followed by B. gymnorhiza (−13.16 ± 2.86%), B. cylindrica (−10.7 ± 1.97%), C. tagal (−20.21 ± 0.4%), K. candel (−38.24 ± 3.64%), and R. mucronata (−25.26 ± 3.29%). A negative value means the wound area is increasing rather than decreasing in comparison to the control cells (untreated cells), meaning the cells are not migrating into the wound, but rather experiencing detachment, apoptosis or inability to migrate [35]. The images showed clear variations in cellular activity between the groups. Cells were visibly rounded, and a drastic reduction in cellular motility was observed leading to aberrant cellular behaviour, in contrast to the control group, where the cells maintained their typical cellular morphology intact, thereby migrating in a coordinated and uniform manner [36]. Hence, we can conclude that the cytoskeleton was probably disrupted by the methanolic extract treatment. Results obtained from the wound healing assay clearly suggest the antimigratory effect on the cell lines exerted by the methanolic mangrove extracts at a concentration of their IC50 values.

LC-HRMS profiling revealed the presence of 107 distinct metabolites
In the present study, a sensitive and high-throughput method of liquid chromatography-high-resolution mass spectrometry was used to identify the chemical constituents in the methanolic leaf extracts of the selected mangrove plants. The chromatograms for all the extracts are shown in Supplementary Figures 2-7. The qualitative analysis of the LC-HRMS data of the phytochemicals across the six mangrove species revealed notable variations in the relative composition of secondary metabolites, which highlight their diversity in phytochemical composition and distinct metabolic adaptations. The acquired MS data were processed using standard mzCloud and ChemSpider database. Among the various identified bioactive molecules, the phytochemicals were categorized into flavonoids, fatty acids, terpenes, carboxylic acids, phenols, alkaloids, and others (encompassing minor constituents) classes of secondary metabolites. The molecular masses, retention indexes, and spectral properties of all the mass signals were compared to the chemical and spectral databases. If the MS analysis comprising of ionization ratios, mass peak, and fragment peaks were dissimilar to the previously reported data, the matches were excluded and classified as unidentified. Only those compounds matching all reported parameters are included in Tables 4, 5, 6, 7, 8 and 9. A total of 44 diverse chemical constituents were identified from the methanolic extract of B. cylindrica, 32 from R. apiculata, 51 from K. candel, 55 from B. gymnorhiza, 46 from C. tagal, and 24 from R. mucronata. Many noteworthy compounds were identified that may contribute to the anticancer properties of the mangrove extracts, namely kaempferol, luteolin, quercetin, nobiletin, and myricetin. The structures of the identified compounds with potent anticancer properties is shown in Fig. 6. The class of compounds such as carboxylic acids and fatty acids gets easily deprotonated and detected in negative ion mode, whereas the class of compounds such as terpenoids, flavonoids, and fatty acids respond in positive ion mode. The genus Rhizophora exhibited the highest flavonoid content (24.1% in R. mucronata and 27.3% in R. apiculata). R. apiculata (15.2%) and C. tagal (18.8%) showed maximum abundance of phenols. Interestingly, alkaloids were detected only in four species, with B. cylindrica showing the highest relative abundance (4.7%) (Fig. 7).

Discussion
Mangrove ecosystems are exposed to extreme environmental conditions such as high salinity, intense UV radiation, tidal fluctuations, and anoxic soils, which collectively induce strong metabolic adaptations. These abiotic stresses activate the secondary metabolite biosynthesis, which results in the production of a diverse array of bioactive compounds, which includes phenolics, flavonoids, terpenoids, and alkaloids. These bioactive compounds contribute to stress tolerance and defense mechanisms in these plants. In addition, they also exhibit remarkable anticancer, antioxidant, and anti-inflammatory activities [1–3]. Thus, these unique abiotic stresses experienced by mangrove habitats are key drivers of secondary metabolite diversity, reinforcing the potent anticancer potential demonstrated by their extracts in our study. The purpose of this study was to comprehensively examine the anticancer potential inherent in the Rhizophoraceae family specifically focusing on the bioactive compounds present within their leaves. Rhizophoraceae is one of the most dominant and widely distributed mangrove family across the Indo-Pacific region and have been traditionally recognised for various ethnomedicinal uses, yet their anticancer potential remains comparatively underexplored and undocumented [4, 6, 8]. The methanolic leaf extracts of the mangroves of the representative genus (Rhizophora, Bruguiera, Ceriops and Kandelia) of the family Rhizophoraceae namely R. mucronata, R. apiculata, B. gymnorhiza, B. cylindrica, C. tagal and K. candel were examined for their anticancer activity using MTT assay. MTT assay was performed on multiple cancer cell lines, namely AW13516 (oral squamous carcinoma), A549 (non-small cell lung carcinoma) and HeLa (cervical cancer) in order to comparatively evaluate the cell type specificity and general cytotoxic potential of the methanolic leaf extracts of the selected mangrove species. This strategic selection of multiple cell lines aided us to evaluate the anticancer activity of the mangrove extracts across different cancer cell types. Additionally, the HaCaT (human keratinocyte) cell line was included to ensure that the observed cytotoxicity was preferentially directed toward cancer cells rather than normal human cells. By employing MTT assay we determined the IC50 values of each extract for the cancer cells under study. The IC₅₀ value represents the concentration of an extract or a compound required to inhibit 50% of cell viability in comparison to the untreated cells. It is a critical parameter in cytotoxicity assays as it quantitatively reflects the potency and efficacy of a test sample against cancer cells. A higher cytotoxic potential is indicated by a lower IC₅₀ value, whereas lower cytotoxicity is indicated by a higher IC₅₀ value [20]. Therefore, determining IC₅₀ values allowed us to perform the comparative assessment of cytotoxic strength of the methanolic extracts of the selected mangrove species which further helped us in identifying the mangrove species with the most promising anticancer activity. In addition, the selectivity was assessed by calculating the SI value for each mangrove extract corresponding to each cancer cell line. Selectivity Index (SI) is a crucial parameter for the evaluation of the therapeutic safety and specificity of anticancer agents. A higher SI value indicates that the extract is more toxic to cancer cells while sparing the normal cells, thereby, reducing the risk of systemic toxicity. Non-selective cytotoxicity often leads to severe side effects and limits clinical applicability [30]. Furthermore, our study concurrently evaluated the anticellular migratory effects of the extracts on cancerous cell lines using in-vitro scratch wound healing assay on A549 cells.
Evaluation of both cytotoxicity and cell migration is important for a comprehensive assessment of anticancer agents. On one hand cytotoxicity assays reveal a compound’s ability to inhibit proliferation or induce cell death, while migration assays, such as the scratch wound healing assay, assess its anti-metastatic potential. Since metastasis depends on cancer cell movement and invasion, combining these assays offers a broader understanding of therapeutic efficacy in suppressing tumor growth and preventing cancer spread [20, 30].
Phytochemical analysis employing liquid chromatography high resolution mass spectrometry (LC- HRMS) enabled the identification of several noteworthy metabolites that significantly contribute to the observed anticancer and anti-migratory properties associated with the mangrove leaf extracts. The acquired MS data was processed using standard mzCloud and ChemSpider database. It was of utmost importance to compare the molecular masses and retention indices with spectral databases for the accurate identification of metabolites. By matching these parameters with reference data in spectral databases, the reliability and confidence of compound identification was significantly increased. This comparative approach helped us to differentiate closely related compounds ensuring that the detected metabolites were correctly annotated and biologically relevant to the observed activities.
Recent studies on the anticancer properties of mangrove-derived extracts from diverse plant parts has yielded some intriguing outcomes. Cytotoxicity effects of the B. gymnorhiza’s methanol, diethyl ether, and butanol extracts on the MCF-7 cell line showed potent cytotoxic activities in a dose dependent manner [94]. Phytosterols extracted from the root bark of R. apiculata exhibited significant anticancer activity against MCF-7, A549, and HeLa cancer cell lines [95]. The fruit methanol extract of the mangrove C. tagal exhibited significant cytotoxicity against MDA-MB-231 (breast cancer) and HCT-116 (colon cancer) cell lines with IC50 values of 50.57 µg/mL and 38.51 µg/mL respectively [96]. A limited body of literature and research findings exists on the anticancer properties of methanolic leaf extracts from Rhizophoraceae mangroves, specifically concerning human lung and cervical cancer cell lines, and no studies have investigated their efficacy against oral cancer cell lines. The MTT assay serves as a tool for evaluating the inherent cytotoxic effects of plant extracts, thereby facilitating the establishment of a permissible dosage concentration for administration [20]. Our findings from the MTT assay and the SI index indicated that among all tested species, B. cylindrica exhibited a pronounced IC50 value, characterized by a minimum half-maximal inhibitory concentration of 287.062 ± 1.127, 323.965 ± 1.417 and 25.942 ± 2.048 µg/mL and SI values of 5.60, 4.96 and 62.03 against AW 13516 (oral), A549 (lung) and HeLa (cervical) cancer cell lines respectively. Among these, the HeLa cell lines showed the highest susceptibility, underscored by the lowest IC₅₀ value. This indicates strong inhibition of cervical cancer cell proliferation. The AW 13516 and A549 cancer cells also demonstrated substantial cytotoxic effects, though with slightly higher IC50 values compared to HeLa cells.
The above data collectively signifies that B. cylindrica exhibited the best therapeutic index (high toxicity towards cancer cells and low toxicity towards normal cells). Overall, the observed SI values reinforce the notion that all of the mangrove species under study possess bioactive compounds capable of discriminating between cancer and normal cells, thereby supporting the hypothesis that the mangrove extracts under study exhibit targeted cytotoxicity.
Doxorubicin (DOX) has been used as a reference standard drug (positive control) since it is widely used clinically and is a non-selective anticancer drug. Comparing the IC50 values of the methanolic leaf extracts of the mangroves with those of doxorubicin provided an essential reference point for evaluating the relative therapeutic potential of the crude extracts. DOX exhibits multifaceted anticancer properties, encompassing mechanisms that include inducing genetic mutations, triggering oxidative stress, promoting programmed cell death, ferroptosis, senescence, autophagy and pyroptosis induction, as well as mediating immune responses. On the other hand, DOX has a deleterious impact on normal cellular physiology, giving rise to a spectrum of potentially life-threatening adverse effects, encompassing nephrotoxicity, cardiotoxicity and myelosuppression [97]. Consequently, a substantial need exists for the identification and exploration of viable alternative strategies in the development of drugs derived from bioactive compounds that can serve as efficacious leads in cancer treatment.
The anticancer efficacy of B. gymnorhiza and R. mucronata is substantiated by the results obtained from the MTT assay and selectivity indexes, signifying their potential candidature in anticancer drug development. The IC50 values of B. gymnorhiza and R. mucronata against A549 cells were recorded to be 400.226 ± 1.488 and 399.05 ± 1.255 µg/mL respectively with a selective index of 5.39 and 4.98 respectively. A separate investigation yielded similar outcomes, wherein the IC50 value was determined to be 376 ± 9 μg/mL against A549 cell lines following administration of methanolic R. mucronata (sample collected from mangrove vegetation near Shalateen city in Egypt) extract. The methanolic leaf extract of R. mucronata exhibited IC50 value of approximately 376 ± 9 μg/mL against A549 cell lines [16]. Notably, the same study demonstrated that the methanolic extract from R. mucronata leaves exhibited minimal cytotoxicity against non-cancer cells (WI-38) with IC50 values of 932 ± 30 μg/mL, a phenomenon mirrored in our current study where the methanolic extract of R. mucronata leaves showed no cytotoxic effects against non-cancerous keratinocytes (HaCaT) with an IC50 value of 1987.675 ± 17.079 µg/mL. The scratch wound healing assay is a well-established in vitro method to evaluate the cell migration and wound closure, often offering valuable insights into the antimigratory potential of test compounds. A scratch is created on a confluent cell monolayer, and the rate of closure is observed with and without treatment. As cancer metastasis relies on cell movement of the cancer cells and invasion, mangrove extracts that inhibit or delay cell migration are considered to possess anti-metastatic properties [35]. Thus, this assay provides a simple yet effective model for screening mangrove extracts for potential anti-metastatic activity. The in vitro scratch wound healing assay revealed significant insights into the antimigratory potential of selected mangrove extracts. All methanolic extracts uniformly exhibited negative migration rates, which resulted in an augmented gap area relative to control cells due to potential factors such as cell detachment and cell death. Notably, extracts from R. apiculata, K. candel and R. mucronata demonstrated substantial antimigratory activity on A549 cells, suggesting bioactive compounds within these extracts interfere with cellular processes associated with metastasis, potentially through disruption of cytoskeleton dynamics [35]. Our analysis corroborated the findings of Rezadoost et al. (2019), demonstrating that the rate of cytotoxicity in a variety of cancer cell lines is affected differently by the bioactivity of different plant extracts [98]. While evaluating the cytotoxic effects of the selected mangrove species, disparate outcomes were observed, with certain species demonstrating high cytotoxicity against cancer cells whilst others displayed modest activity. However, none of these species elicited measurable cytotoxicity in normal HaCaT cells, thus conforming to established protocols for non-toxic compounds proposed by Geran and the National Cancer Institute [16]. Based on the criteria used to categorize the cytotoxicity of plant crude extracts against cancer cell lines by U.S. National Cancer Institute (NCI) and Geran protocol, among all the mangrove extracts, B. cylindrica highlighted the highest cytotoxic potential, with an IC₅₀ value of 25.94 µg/mL against HeLa cells, categorizing it as moderately cytotoxic, while its activity against A549 and AW 13516 cells was weakly cytotoxic [30, 99]. Extracts of B. gymnorhiza, R. mucronata, R. apiculata, K. candel and C. tagal demonstrated moderate cytotoxic activity against HeLa cells but weakly cytotoxic against A549 and AW 13516 lines. This dichotomy in activity may be attributed to the unique phytochemical profiles present in each species, as supported by LC-HRMS data.
Dissimilarities in the metabolic compositions amongst the six investigated species, such as the occurrence of alkaloids in K. candel, sesquiterpenes and triterpenoids in B. gymnorhiza, pronounced accumulation of flavonoids within the Rhizophora genus and substantial amounts of phenols in R. apiculata and C. tagal, illustrate the phytochemical diversity influenced by evolutionary and ecological factors.
Flavonoids are widely dispersed in plants and are regarded as crucial in pharmacological, medical, nutraceutical, and cosmetic applications due to their anti-inflammatory, antioxidant, and anti-tumor activities [100]. In the mangrove extracts, they were mostly present in the form of flavones and flavonols such as rutin, quercetin-3β-D-glucoside, quercetin, luteolin, nobiletin, naringenin, and catechin.
Quercetin, a widely studied flavonoid, has been shown to induce apoptosis by decreasing bioenergy, targeting mitochondria, and inhibiting metastasis in breast and prostate cancer cells [101]. In our study, quercetin was ubiquitously found in all the mangrove extracts under study. Similarly, catechin and its derivatives have demonstrated strong radical scavenging activity, contributing to their potential role in cancer prevention and therapy as they have been reported to be critical for the induction of apoptosis and inhibition of cancer cell growth [102]. Catechin was detected in B. gymnorhiza, C. tagal and R. mucronata in our study. Among plants, phenolics are present ubiquitously. The phenolic acid identified in our study includes chlorogenic acid, caffeic acid, gallic acid, and jasmonic acid. These compounds are known to modulate key signaling pathways in cancer progression, including inhibition of NF-κB activation and downregulation of inflammatory mediators and have specific action on the various checkpoints of cancerous cells [103, 104]. Chlorogenic acid was identified as a common constituent across all the mangrove species under study, suggesting its widespread occurrence and possible role in their bioactivity. Caffeic acid was detected exclusively in B. cylindrica and C. tagal, while gallic acid was present in K. candel and B. gymnorhiza. Interestingly, jasmonic acid was found only in K. candel. In relevance to the type of terpenes found in the leaf extracts, majorly sesquiterpenes, triterpenoids, and diterpenoids were found, including betulin, trans-caryophyllene oxide, 18β-Glycyrrhetinic acid, and abscisic acid.
Betulin was detected in B. cylindrica, R. apiculata, K. candel, and C. tagal. Trans-caryophyllene oxide was present in K. candel, C. tagal, B. gymnorhiza, and B. cylindrica. 18β-Glycyrrhetinic acid was identified in all species except R. apiculata.
The proposed mechanism of action underlying the anticancer effects of the mangrove leaf extracts can be attributed to the bioactive flavonoids and phenolic compounds identified in our study. Previous literature has extensively reported the molecular pathways through which these metabolites exert cytotoxic, pro-apoptotic, and anti-migratory activities.
Naringenin, a flavonone detected in the crude methanolic extracts of R. apiculata, K. candel, C. tagal, and R. mucronata, has been shown to induce programmed cell death in SGC-7901 cells via upregulating key pro-apoptotic proteins, including caspases-3, p53, and BAX proteins, and simultaneously downregulating anti-apoptotic proteins Bcl-2 and Survivin [105, 106]. These cellular processes contribute to initiation of the extrinsic apoptosis pathway, further supported by the elevated expression of TNF-family proteins [106]. Luteolin, a flavone compound present in R. mucronata, R. apiculata, B. gymnorhiza, C. tagal and B. cylindrica modulates Akt, JNK, and p38 signaling pathways by initiating autophagy and triggering apoptosis in MCF-7, ANA-1, and gastric cancer cell [107]. The decreased expression of Bcl-2 and BECLIN1, along with elevated levels of caspase-3 and caspase-8 marks apoptotic induction [102]. Quercetin, a flavonol, inhibits cell cycle and initiates apoptosis [101, 108]. Kaempferol, another flavonol, found in 3 extracts under study, namely, R. apiculata, K. candel and B. gymnorhiza, helps initiate apoptosis and induction of autophagy via increased expression of miR-340 microRNA in (Colon) HCT-116, HCT15, SW480, and A549 (human lung cancer) cell lines [109]. Myricetin, a flavonol detected in R. apiculata and B. gymnorhiza, inhibits metastasis by inhibiting cell migration as seen in prostate cancer [65, 110]. Taxifolin, a flavonol found in C. tagal and B. gymnorhiza, inhibits carcinogenesis through mTOR/PTEN axis and CYP1B1 mediated cancer [111]. Catechin, which is a flavan, decreases cancer growth through programmed cell death [112]. The varied bioactive flavonoids and phenolic compounds present in the tested mangrove extracts are likely responsible for the observed anticancer activity. Bioactive compounds such as naringenin, luteolin, quercetin, kaempferol, myricetin, taxifolin, and catechin, which have been extensively characterised for their roles in modulating apoptosis, autophagy, cell-cycle arrest, and metastasis inhibition, contribute to the crude extracts' cytotoxic effects. The observed anticancer activity of the mangrove extracts may be attributed not only to individual bioactive compounds but also to possible synergistic interactions among multiple metabolites. The cytotoxic potential is probably enhanced by the synergism by simultaneously targeting different molecular pathways, including induction of apoptosis, modulation of oxidative stress, and inhibition of cell proliferation or migration. Compounds such as flavonoids, phenolic acids, terpenoids, and alkaloids may act additively, amplifying their overall therapeutic effect compared to their isolated forms [113]. In our study, several methanolic crude extracts of selected mangrove species exhibited moderate to weak cytotoxicity against different cancer cell lines based on U.S. National Cancer Institute (NCI) and Geran protocol [16]. In particular, B. cylindrica displayed strong activity against HeLa (cervical cancer) cells with an IC₅₀ value of 25.942 ± 2.048 µg/mL, placing it at the borderline of the highly cytotoxic range. Notably, the highest selectivity index (SI) of 62.03 was observed for B. cylindrica against HeLa cell lines, underlining the preferential cytotoxicity towards cervical cancer cells while sparing the normal human keratinocyte cells. This selective cytotoxicity is a critical property in the development of anticancer drugs, serving to mitigate adverse effects on normal cells by ensuring preferential targeting of malignant cells. This underscores its potential as a promising anticancer candidate.
Some compounds previously reported from the mangrove species did not appear in our LC-HRMS analysis. One explanation for the above result could be that some compounds are better extracted using other solvents like ethyl acetate, ethanol, acetone, or even water, while others that are poorly soluble in methanol might have escaped our phytochemical screening. Other reasons include different seasons of sample collection, polarity in the geographical conditions, and experimental methods. Thus, our results could prove important for the selected mangrove species as some compounds were reported for the first time.
In our present study, we used crude methanolic leaf extracts of selected mangrove species, namely, R. mucronata, R. apiculata, B. gymnorhiza, B. cylindrica, C. tagal, and K. candel. These crude extracts provided an important preliminary screening for evaluating their cytotoxic activities, which helped us highlight the most promising species, but also present certain limitations. As confirmed by LC-HRMS analysis results, crude extracts contain a complex mixture of phytochemicals, making it difficult to attribute the observed biological activity to specific bioactive constituents. Additionally, potential synergistic or antagonistic interactions among the compounds may influence the measured cytotoxic effects [114]. In order to overcome these limitations, future studies should be focused on bioassay-guided fractionation and isolation of active compounds using chromatographic techniques such as thin layer chromatography (TLC), column chromatography and high performance liquid chromatography (HPLC) followed by structural characterization of the bioactive compound(s) using fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR). To confirm the mechanisms of action, pharmacokinetics, and safety profiles of the purified compounds, target validation studies and in vivo evaluations should be conducted. This would help elucidate the precise molecular targets responsible for the observed anticancer and anti-migratory properties of the bioactive compounds.
Nonetheless, the presence of the diverse array of bioactive compounds underscores the potential of the mangrove extracts as a natural source of anticancer agents and could be used as a potential alternative for the development of bioactive leads in the treatment of cancer.

Conclusion
In the findings of our present study, the methanolic leaf extracts from six mangrove species in the Rhizophoraceae family highlighted significant anticancer potential, particularly in B. cylindrica. In addition to this, the methanolic leaf extracts successfully inhibited cancer cell migration. Flavonoids, terpenoids, phenolic acids, carboxylic acids, fatty acids and alkaloids were the major classes of the phytochemicals that were identified, which have been previously known to modulate key oncogenic pathways in the cancer cells, likely contributing to the observed cytotoxicity and migratory inhibition. Bioactive compounds from mangroves have proven to be a valuable source of novel chemotherapeutic agents, having tremendous potential to be explored for its synergistic formulations and drug delivery systems to enhance therapeutic efficacy and pave the way for the clinical translation of mangrove-derived phytochemicals in cancer treatment. Further research has to be carried out in order to elucidate the molecular mechanisms underlying their anticancer effects, validating their efficacy in in vivo models, and optimizing their bioavailability for clinical applications.

Supplementary Information