Investigating the Medicinal Potential of Lavatera cashmeriana Leaf Extract: Phytochemical Profiling and In Vitro Evaluation of Antimicrobial, Antioxidant, and Anticancer Activities

This study investigated the medicinal potential of Lavatera cashmeriana, a plant traditionally known for its therapeutic properties. The aim was to identify the phytocompounds in L. cashmeriana leaf extract and evaluate its antibacterial, antioxidant, and anticancer effects. Gas chromatography-mass spectrometry analysis was employed to characterize the phytochemical composition of the ethanol extract derived from L. cashmeriana leaves. The antimicrobial potential was assessed through the well diffusion technique, targeting Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. The 2,2-diphenyl-1-picrylhydrazyl assay was conducted to assess antioxidant capabilities, while cytotoxicity against the A549 cancer cell line was determined via the MTT assay. GC-MS analysis identified ten different compounds, with phytol, 1-Eicosanol, and 2,6,10-trimethyl,14-ethylene-14-pentadecne being the most prevalent. The extract exhibited notable antimicrobial efficacy against all bacteria with MIC values ranging from 62.5 to 250 µg/mL. However, C. albicans did not respond. The extract exhibited antioxidative properties with an IC50 value of 86 µg/mL and cytotoxicity with an IC50 value of 69.95 µg/mL against the A549 cancer cell line. The results derived from this study supported the historical use of L. cashmeriana as a medicinal plant and suggested that it can potentially treat a wide range of medical ailments. The identified phytocompounds and the demonstrated antibacterial, antioxidant, and anticancer effects provide scientific evidence for its medicinal properties. However, further investigations are needed to fully understand its safety profile, efficacy, and mechanism of action before recommending it for therapeutic purposes.


Introduction
Plants have long been utilized in the treatment and alleviation of numerous illnesses.Despite the efcacy of synthetic drugs in addressing various diseases, herbal remedies are often preferred due to their fewer side efects and greater accessibility [1].Plants contain secondary metabolites that exhibit diverse biological activities, such as antidiabetic, anticancer, antibacterial, anti-infammatory, and antioxidant properties [2].Gaining insight into the chemical makeup of plants is of utmost importance, as this knowledge can contribute to developing novel bioactive compounds tailored to target specifc diseases [3].Phytochemicals derived from medicinal plants are crucial as primary ingredients in the development and discovery of pharmaceuticals [4].Te primary healthcare needs of more than 80% of the global population are met through the use of plant-based remedies, as stated by the WHO [5].
Antimicrobial drug resistance has been a global concern in recent years since resistant microbes cause many health issues due to their genetic plasticity [6].Te most common pathogenic bacteria are S. aureus, P. aeruginosa, K. pneumoniae, S. typhimurium, S. epidermidis, E. coli, and C. albicans, which have been linked to antimicrobial resistance [7].
Research on relationships between plant-based antioxidants and the prevention of noncommunicable diseases, like diabetes, cancer, and cardiovascular, has increased sharply in recent years [8].Epidemiological and in vitro investigations provide strong evidence supporting the potential use of plants containing antioxidant phytochemicals to treat various ailments.Phytochemicals, mainly phenolic compounds, have been shown to reduce oxidative stress and inhibit macromolecular oxidation, thereby decreasing the risk of degenerative diseases [9].Consequently, there is a growing interest in identifying plant extracts that exhibit efective and nontoxic properties.
In the present era, cancer continues to be one of the most formidable illnesses, posing an imminent danger to human life.Numerous internal and external factors contribute to the onset of this disease.Despite recent strides in the development of novel anticancer medications, cancer remains a leading cause of mortality on a global scale [10].Te emergence of resistance to chemotherapy compounds further complicates the efectiveness of cancer treatment, serving as a signifcant obstacle.Consequently, the exploration and identifcation of fresh, efcacious anticancer drugs to counteract this resistance have become imperative challenges [11].Over the past few decades, natural products have signifcantly contributed in the development of chemotherapeutic drugs.A noteworthy proportion of successful cancer treatments owe their success to compounds sourced from nature [10].Approximately, 60% of the drugs presently employed in cancer treatment trace their origins back to natural sources [12].
Lavatera cashmeriana is an important medicinal herb of the Kashmir Himalayas that belongs to the Malvaceae family.Despite being native to the Kashmir valley, it is now widespread throughout the western Himalayas, from Pakistan to Uttar Pradesh and Uttaranchal.L. cashmeriana roots have been traditionally employed to address various health issues, including gastrointestinal problems, kidney stones, and dandruf.Tey serve as a laxative and are believed to stimulate hair growth when applied to the scalp.Traditionally, the roots of L. cashmeriana are used to treat gastrointestinal diseases, renal colic, and dandruf.Te root decoction of L. cashmeriana is used as a laxative and is also said to promote hair growth when applied to the scalp.Its fowers are used to treat common colds and mumps, and its seeds are used as an antiseptic [13,14].Te leaves and petals have been used to treat skin irritation in pregnant women, urinary issues, and as an antiseptic [15][16][17].Recent research on this species has revealed some biological activities, including antilipoxygenase activity [18] as a protease inhibitor [19].Crude extracts of medicinal plants are believed to possess higher biological activity than isolated phytocompounds due to their synergistic efects [20].Te synthesis of these secondary metabolites is genetically controlled and is strongly infuenced by various biotic and abiotic stresses.Various environmental factors, such as precipitation, mean temperature, soil characteristics, and radiation, change with altitude [21,22].Te present study aims to identify chemical constituents and assess the antimicrobial, anticancer, and antioxidant properties of the ethanolic leaf extract of L. cashmeriana.

Plant Collection and Processing.
Te plant depicted in Figure 1, L. cashmeriana, was procured from Daksum Anantnag, Jammu and Kashmir, India, in June 2019.Tis region is situated at an elevation of 2,438 meters (Figure 2).Te plant was identifed and authenticated at the Kashmir University's Center for Biodiversity and Taxonomy (CBTbotany) under voucher 3070-(KASH) Herbarium.Te plant material (leaves) was subjected to a shade-drying process under hygienic conditions for a minimum of 15 days to facilitate further processing.Subsequently, an electrical grinder was used to crush the dried leaves into a coarse powder form.Te resulting powder was then packaged carefully to maintain its quality and purity for future use in research.

Extraction Process.
Te extraction of the plant material was performed using a simple maceration process [1].A total of 10 grams of coarse powder from the plant material was combined with 200 mL of ethanol, of a desired quality grade, in a fask.Te mixture was then placed on a shaker and underwent extraction for 24 h at room temperature.After 24 h, the reaction mixture was fltered using Whatman flter paper No. 1. Te obtained fltrate was further processed through solvent evaporation to get a more concentrated extract.Tis process was performed thrice to ensure the sample's sufcient quantity and quality for subsequent analysis.
Total extraction yield % � mass of the sample mass of the extract   × 100. (1)

GC-MS Analysis
Phytochemical profling analysis was conducted using the GC-MS Shimadzu-QP2010.Te extract was introduced to a capillary column through split mode injection with split ratio of 1 : 2. Te carrier gas used was helium, and the fow rate was set at 1 mL per minute.Te analysis spanned 50 minutes.Initially, the column oven was set at 80 °C.Subsequently, the column temperature was gradually increased by 3 °C per minute until it reached 200 °C.Following this, the temperature was further elevated to 260 °C at a rate of 10 °C per minute and held steady for 5 minutes at that level [23].

List of Microbial Species
Table 1 represents the microbial species employed in the antimicrobial assay screening.

Antimicrobial Assay
We evaluated the antimicrobial properties of the methanolic leaf extract from L. cashmeriana against diferent microbes (Table 1) using the well difusion method.Initially, sterile Muller Hinton Agar was poured into Petri dishes and allowed to solidify.Subsequently, test microorganisms were uniformly spread over the agar surface at densities ranging from 10 4 to 10 6 CFU/mL.Wells were created on the agar surface using a cork borer, and four wells were flled with varying concentrations of the L. cashmeriana extract ranging from 25 to 100 μL, each from a 10 mg/mL stock solution.Te ffth and sixth wells served as the positive and negative controls, respectively.Following incubation at 37 °C for 16-18 h, zones of inhibition were measured around the wells.

Determination of the MIC.
Te procedure was executed within 96-well plates, following the methodology outlined by Gabrielson et al. [24].Te initial ten wells of the plate accommodated diverse concentrations of L. cashmeriana leaf extract, ranging from 500 to 0.9 µg/mL.Once the wells were loaded with the extract, 5 µL of 12 h old microbial culture was introduced to each well.Subsequently, the plates were positioned within an incubator set to 37 °C, where they remained for a duration of 16-18 h.Subsequent to the incubation, 10 µL of a 5 mg/mL MTT solution was

Antioxidant Activity
Te radical scavenging activity (RSA) of the sample was evaluated using the DPPH assay, following the methodology outlined by Chang et al. [25] with minor adjustments.In this procedure, a 0.1 mM ethanolic DPPH solution (2960 µL) was mixed with varying concentrations of the plant extract (20,40,60,80, and 100 µg), alongside a blank solution composed of 0.1 mM ethanolic DPPH solution (40 µL) mixed with distilled water.Te solution was vigorously mixed using a vortex mixer and afterwards placed in a dark environment at room temperature for a period of 20 minutes.Subsequently, the absorption of the mixtures was recorded at 517 nm.As a reference compound, BHT was employed.Te RSA was computed using the subsequent formula: RSA (%) � (absorbance control − absorbance sample) × 100.

Cell Culture
Te A549 human lung cancer cell line was obtained from NCCS Pune, India.Tese cells were cultured in DMEM (Sigma-Aldrich, United States, D1152) supplemented with 10% FBS (Gibco, United States, 10270106) and 1% antibioticantimycotic solution.Tey were maintained in an incubator at 37 °C with 5% CO 2 .Te cells were allowed to grow until they reached 70-80% confuency, indicating that they had formed a monolayer on the surface of the culture dish.Trypsinization was performed using 1× trypsin EDTA (Gibco, Catalogue: 25200-056) solution to propagate the cells further.Following trypsinization, the A549 cells were seeded into a 96-well plate and given 21-24 h to attach to the surface.

MTT Assay.
In this experiment, viable cells were cultured in a 96-well plate, with each well containing a density of 6,000 cells.Extracts were prepared at various concentrations (6.5, 12.5, 25, 75, 100, and 125 μg/mL) using a culture medium.Tis treatment was administered over 24 and 48 h.Following the treatment period, the medium containing the extract was carefully removed and substituted with a new serum-free medium.After incubation, the MTT reagent was introduced into each well and allowed to incubate for another 4 h.Te formation of formazan crystals, indicating viable cells, was facilitated by the MTT reagent.Subsequently, these crystals were solubilized using DMSO, and the absorbance was determined at 570 nm [26].Te % of cell inhibition was calculated employing the following equation: % of viability � absorbance of control cells absorbance of treated cells   × 100.(2) 7.2.Morphological Analysis.Cells were seeded onto glass coverslips in a 6-wellplate and allowed to grow until they reached a healthy morphology and achieved 60-80% confuence.Once this stage was reached, the cells were exposed to specifc IC 50 concentrations of L. cashmeriana leaf extract and incubated for 24 h.Upon completion of the experiment, the cells were rinsed twice with PBS.Morphological analysis was then performed using an inverted microscope (Motic type 101M).

Statistical Analysis
Te experiments were carried out, and the data were analyzed to compute the mean ± standard deviation and IC 50 values for both DPPH and MTT assays using Microsoft Excel 2019.

Results and Discussion
Te percentage of plant extract obtained using ethanol as a solvent was assessed, and the outcomes are presented in Table 2.
Te GC-MS study of the L. cashmeriana ethanolic leaf extract resulted in the identifcation of ten compounds (Figure 3 and Table 3).
Of these phytocompounds, the most prevailing compounds were phytol, 2,6,10-trimethyl,14-ethylene-14-pentadecane, and 1-Eicosanol.Phytol is the most prevalent and identifed compound in the leaf extract of L. cashmeriana.Phytol has been investigated for its health benefts, including its ability to decrease blood sugar levels, reduce infammation, and improve liver function.It is also commonly used in the fragrance and favor industry and the production of vitamins and other supplements.It has also been shown to have potential as an antimicrobial, anticancer agent, as it has been found to induce apoptosis in cancer cells [27].
Te emergence of antibiotic resistance and new pathogens has made treating bacterial infections more challenging.Terefore, fnding new and efective bioactive agents to combat microbial infections is urgently needed.Despite the availability of numerous drugs for treating microbial infections, their adverse side efects can limit their usage in specifc populations, making it difcult to treat microbial infections, especially in vulnerable populations [28,29].Plant extracts are active against various pathogenic microorganisms, including S. typhi, E. coli, P. aeruginosa, C. albicans, S. aureus, and B. subtilis, among others.As a result, researchers continue to explore the potential of plant extracts as a source of new and efective bioactive agents to treat bacterial infections [30].L. cashmeriana, a medicinal plant commonly used in traditional remedies by local inhabitants, was tested against diferent microbial strains to evaluate and examine its antimicrobial capabilities.Te fndings of this study are encouraging, as the examined microbes commonly cause community-and hospital-acquired infections.Te growing antibiotic resistance underscores the need for new drugs with novel targets.Te antimicrobial activity of L. cashmeriana is relevant in this perspective since the MIC values of the active extract in the present investigation are below 1 mg/mL, which may be regarded as signifcant in light of the discovery of potent antimicrobials from plants.
Te generation of free radicals causes several reactions that may harm tissues, cell function, and macromolecules [36].Oxidative stress has been linked to several pathological disorders, including aging, atherosclerosis, diabetes, and cancer [37].Nonetheless, antioxidants provide a promising defense against oxidative stress-related diseases by slowing the rate of oxidation or inhibiting the propagation of free radical production, resulting in anticancer, anti-aging, and anti-infammatory properties [38].Te antioxidant property of crude extract was assessed using the DPPH radical scavenging test in this study.Te fndings of this assay are shown in Figure 5, which compared the DPPH radical scavenging activity of L. cashmeriana leaf extract to that of ascorbic acid.Although the standard antioxidant had more scavenging activity than the extract at all tested concentrations, the extract nonetheless demonstrated considerable free radical scavenging activity.One of the methods through which L. cashmeriana is benefcial as traditional medicine is its ability to scavenge free radicals.Consumption of L. cashmeriana leaves may help in preventing oxidative stress-related degenerative illnesses.Te antioxidant activity of L. cashmeriana might be due to the presence of favonoid and phenolic compounds.Phenolic and favonoid compounds have been linked to antioxidative activity in biological systems, serving as scavengers of free radicals [39].Consumption of L. cashmeriana leaves may help in preventing oxidative stress-related degenerative illnesses.
Cancer has become a signifcant cause of mortality in underdeveloped nations, surpassing AIDS, TB, and malaria combined.Lung cancer is a particularly prevalent type, leading to a large number of deaths worldwide [40,41].Although synthetic chemistry has recently dominated drug discovery and production, plant-derived medications have signifcantly impacted the antitumor feld.In light of this, the potential of bioactive plants and their extracts to provide innovative disease treatment and prevention solutions remains immense.Some of the notable plant-derived agents that have improved chemotherapy for various types of cancer include taxol, vinblastine, camptothecin, and vincristine [42].Te vast potential of plants to produce chemicals attracts researchers seeking new and novel chemotherapeutic agents [43].As a result, exploring anticancer chemicals in plants and traditional foods is a promising and viable cancer prevention strategy [44].Plantbased compounds with anticancer properties can be found in several categories, such as terpenoids, phenylpropanoids, and alkaloids [45].Continued research into these compounds holds excellent potential for the development of new, efective treatments for cancer, especially in underdeveloped nations where access to conventional therapies might be limited.
Harnessing the power of plants and their extracts can revolutionize cancer treatment and prevention, ofering hope to millions of patients worldwide.Many plant-derived medicines are efective in treating diferent types of cancer.Plant-derived medicines often have advantages, including better patient tolerance and fewer negative side efects in comparison to their synthetic counterparts.In addition, the development of drug resistance is less common with plantderived medications [43,46].
In the past, extracts and isolated components derived from plants belonging to the Malvaceae family have shown noteworthy efectiveness in inhibiting the growth of cancerous cells.Tis current investigation focused on evaluating the impact of an ethanol-based leaf extract from L. cashmeriana on A549 cells.Te extract was found to be cytotoxic to A549 cells, with an IC 50 value of 69.95 µg/mL compared to control (Figure 6).Te cytotoxicity increased with an increase in extract concentration, as observed in the MTT results.Te study suggests that the ethanolic extract of L. cashmeriana leaves showed strong anticancer activity against A549 cells in a dose-dependent manner.Tus, it has the potential for use in cancer prevention and chemotherapy.Earlier research has documented the in vitro potential of extracts from the same family to exhibit antilung cancer properties.Rakshanda et al. [47] studied the impact of L. cashmeriana protease inhibitors (LC-pi) isolated from      [48].Similarly, the ethanolic leaf extract of Abutilon indicum L exhibited antiproliferative efects against the A549 cells, with an IC 50 value of 85.2 µg/mL [49].In another study, the antiproliferative efcacy of the methanolic extract of diferent parts of Grewia orbiculata plant was assessed on the A549 lung cancer cell line, revealing an IC 50 value of 98.73 µg/mL [50].Furthermore, the ethanolic leaf extract derived from A. indicum demonstrated notable antiproliferative efects against the A549 cell line, achieving a substantial cell inhibition rate of 72.1% at a concentration of 200 μg/mL [51].Our fndings align with these earlier studies, suggesting that the ethanolic extract could be a promising candidate for further research and development of anti lung cancer therapies.While preliminary studies have shown promising anticancer properties of L. cashmeriana, further research is essential to validate these fndings and explore the underlying molecular mechanisms responsible for its anticancer efects.Te impact of L. cashmeriana extract on the structure of A549 cells was investigated.In the control group, cells displayed regular growth patterns with distinct nuclei.Furthermore, control cells exhibited excellent adherence in a single layer, whereas the treated cells displayed atypical structure and distorted cell membranes, as evidenced by microscopic analysis (Figures 7(a) and 7(b)).Tese treated cells demonstrated signs of shrinkage and distorted membranes, consistent with the attributes of cells undergoing apoptosis, as previously documented by researchers [52,53].
Phytol, the predominant compound in the ethanolic extract of L. cashmeriana, has been shown to hinder angiogenesis and enhance cell death in A549 cells by altering the mitochondrial membrane potential [61,62].In a separate investigation, it was observed that phytol triggered both apoptosis and protective autophagy in AGS cells.Te autophagy inhibitor CQ intensifed phytol's inhibitory impact on cell proliferation and facilitated apoptosis in gastric cancer cells [63].Likewise, phytol induces apoptotic alterations and generates ROS in A549 cells by activating TRAIL, FAS, and TNF-α receptors, along with caspase 9 and 3 [64].Phytol has been identifed as an inducer of ROS-mediated apoptosis in S. pombe.In addition, it has been confrmed that phytol efectively inhibits cellular senescence caused by oxidative stress from H 2 O 2 [65,66].
Torough investigations of the above analytical compounds present in this plant reveal tremendous medical signifcance, owing to the presence of varied phytoconstituents, which can treat various diseases and potentially serve as precursor compounds for numerous medicinal and other applications.

Conclusion
In conclusion, this research article highlighted the medicinal potential of L. cashmeriana, a plant traditionally used for its therapeutic properties.Te study identifed ten compounds in the ethanol extract of L. cashmeriana leaves, with phytol, 1-Eicosanol, 2,6,10-trimethyl, and 14-ethylene-14-pentadecane being the most prevalent.Te extract exhibited antimicrobial efcacy against E. coli, E. faecalis, P. aeruginosa, and S. aureus.However, it did not afect C. albicans.In addition, the extract showed antioxidant properties and a strong cytotoxic efect on the A549 cancer cell line.Tese positive outcomes necessitate further research to elucidate the mechanisms behind these efects, including the safety and efcacy of the extract in vivo, as well as the exploration of novel therapeutic agents from its active components for the treatment of infectious diseases and cancer.

%Figure 5 :Figure 6 :
Figure 5: Te capacity of ethanolic leaf extract of L. cashmeriana and ascorbic acid to neutralize DPPH radicals is depicted at various concentrations.Each value displayed represents the mean ± SD, based on three repetitions (n � 3).

Table 1 :
Te microbial species employed in the antimicrobial assay screening.Advances in Pharmacological and Pharmaceutical Sciences incorporated into each well.MTT, a yellow dye, is transformed into a purple formazan product exclusively by viable cells.Following 2 h incubation alongside MTT, 100 µL of DMSO was introduced into each well to facilitate the dissolution of the formazan product.Te visual observation of the transition in color, shifting from yellow to purple, enabled the determination of the MIC, an indicator of the lowest extract concentration entirely halting microbial proliferation.

Table 2 :
Te yield of extraction of L. cashmeriana in ethanol.

Table 3 :
Phytocompounds reported in the ethanolic leaf extract of L. cashmeriana by GC-MS.

Table 4 :
Te zone of inhibition of the ethanolic leaf extract of L. cashmeriana against microorganisms.Te symbol -denotes no activity, and the reported value represents the mean ± SD obtained from three repetitions (n � 3).ND: not determined, as they did not show antimicrobial activity in the well difusion assay.