LC-HRMS profiling and in vitro total phenolics, total flavonoids, antioxidant and cytotoxicity evaluation on MCF7 breast cancer cell lines of the extracts and fractions of Aerva sanguinolenta from Java, Indonesia

Sample preparation, extraction, and fractionation of AS

AS, known as SC (AS), was harvested from Tawangmangu, Central Java, Indonesia. As was recognized at their School of Life Science and Technology, ITB Indonesia, under certificate 7881/IT1.C11.2/TA.00/2023. One kilogram of AS plants is cleaned of dirt and dust, dried in an oven (Memmert, Germany) at 40°C–50°C for 24 hours, then ground using a blender (Moulinex, France) until it becomes a fine powder. Next, the extraction is carried out using the maceration method. As much as 500 g of plant powder was placed into the maceration device, and 1 l of technical ethanol (Merck, Germany) was added. The mixture from the maceration is stirred using a magnetic stirrer. After soaking, the mixture is filtered using filter paper to separate the residue from the resulting extract. The filtered extract is then placed into a rotary vacuum evaporator (Büchi, Switzerland) to evaporate the solvent at 40°C and a pressure of 300 mBar, resulting in a thick extract. After the extraction process, the next step is fractionating the obtained methanol extract. As much as 500 g of methanol extract was placed into the fractionation separator (Perekan, Germany). To separate the compounds based on polarity, 200 ml of n-hexane (Merck, Germany) was added to the methanol extract. The mixture was stirred for 10 minutes and allowed to sit for 30 minutes to ensure the fraction separation occurred. After the separation phase, the n-hexane fraction in the upper layer is taken and stored in a storage bottle. The process is done by adding 200 ml of ethyl acetate (Merck, Germany) to the remaining methanol extract, stirring for 10 minutes, and letting it sit for 30 minutes before collecting the ethyl acetate fraction. Next, 200 ml of butanol (Merck, Germany) will be added, and the same process will be performed to obtain the butanol fraction. Finally, the remaining extract is processed by adding 200 ml of water, stirring, and separating it into the aqueous fraction. All the resulting fractions were then evaporated using a rotary vacuum evaporator at the same temperature and pressure to remove the solvent, yielding n-hexane, ethyl acetate, butanol, and water fractions. These fractions are stored in amber glass bottles to maintain stability and quality.

Antioxidant activity: 2,2-dipheinyl-1-picrylhydrazyl free radical scavenging assay

The antioxidant activity was assessed by evaluating the free radical scavenging vcapacity of their AS extract and fraction against their stable 2,2-dipheinyl-1-picrylhydrazyl (DPPH) free radical, following the method established by [16,29] Sarkar et al. [29] and Gulcin and Alwasel [30] with minor changes. 500 mg of their samples and 5 ml of their methanol reaction mixture were combined, vortexed, and sonicated to create a stock solution. Sixteen microliters of each sample were mixed with 1,584 μl of methanol, followed by the addition of 400 μl of DPPH, and their mixture was vortexed. The test samples were generated in triplicate at five concentrations ranging from 62.5 to 1,000 ppm, with their maximum concentration being 1,000 ppm (80 μl + 79,20 μl of methanol). The solution was incubated at an ambient temperature for 30 minutes before measuring their absorbance at a wavelength of 517 nm with a microplate reader (FLUOstar omega). The quantities of several extracts were quantified, and their IC50 value (their concentration necessary to block 50% of DPPH radicals) was established. All assays were conducted in triplicate. Aquadeis served as controls. Meithanoil, combined with DPPH, served as their control. L-(+)-ascorbic acid (Vitamin C) with a final concentration of 0–5 μg/ml was used as the standard antioxidant, measured absorbance value was transformed into the percentage of radical scavenging activity utilizing their subsequent equation [31,32]

$\mathrm{Radical}\mathrm{trapping}\mathrm{activity}\mathrm{(\%)}=\left[\frac{(\mathit{AB}-\mathit{AS})}{\mathit{AB}}\right]\mathrm{x}100\%$

The percentage of radical trapping activity can be determined using their subsequent equation: AB denotes their sample absorption, while AB signifies their absorption of the Blank.

Total flavonoid content

The AlCl3 approach, predicated on the complexation of AlCl3 with flavonoids exhibiting a peak absorbance at 415 nm, was employed to quantify the TFC [33,34]. In summary, 60 μl of ethanol, 5 μl of 10% AlCl3, and 20 μl of each extract (0.5 mg/ml) were mixed separately. After adding 110 μl of distilled water and 5 μl of 1 M potassium acetate each week, their reaction mixture was incubated for 25 minutes. To guarantee experimental repeatability, each measurement was conducted in triplicate. A quercetin calibration curve was employed to determine their TFC; the absorbance was measured at 510 nm. The TFC results are expressed as milligrams of quercetin equivalent (QE) per gram of dry extract weight (mg QE/g) via generating a standard curve with a series of concentrations of 0–100 μg/ml of rutin (x-axis) against absorbance (y-axis).

Total phenolic content

The Folin-Coicalteu method, with slight modifications, was employed to quantify their TPC as described by Cacique et al. [35]. In a 96-well microtiter plate, 20 μl of their sample (0.5 mg/ml) of plant extracts, our standard, and 100 μl of Folin-Coicalteu reagent (1:10; v/v) were added, and their initial absorbance was recorded. The combination was subsequently combined with 80 μl of 1 M Na₂CO₃ solution to achieve a final volume of 200 μl, followed by a 25-minute incubation period. The Synergy LX Multi-Moidei Reader with Gein5 3.08.01 software recorded absorbance at 765 nm. A gallic acid calibration curve was employed to determine their TPC; a standard solution of gallic acid at 1,000 ppm was prepared by dissolving 10 mg of gallic acid in 10 ml of methanol, and a series of standard solutions with concentrations of 300, 250, 200, 150, and 100 ppm were made, results expressed as milligrams of gallic acid equivalent (GAE) per gram of dry extract weight (mg GAE/g). Each measurement was conducted in triplicate for validation purposes.

Cell culture

The MCF-7 cell line, derived from human breast cancer, was procured from the Laboratory for Agro and Industrial Biomedical Technology Development at the National Research and Innovation Agency (BRIN) in Serpong, Tangerang Selatan, Indonesia. It is utilized in the research. MCF-7 cells cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco Life Technologies) media contain 10% fetal bovine serum (FBS), red phenol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. The cells are maintained in a 75 cm² container at 37°C, with 5% CO² and 95% humidity.

Liquid chromatography high-resolution mass spectrophotometry (HRMS) analysis

This analysis utilized liquid chromatography (Thermo ScientificTM VanquishTM UHPLC Binary Pump) in conjunction with Orbitrap HRMS (Thermo ScieintificTM Q ExactiveiTM Hybrid Quadrupoilei-OrbitrapTM HRMS). The liquid chromatography employed a Thermo ScieintificTM AccucoreTM Phenyl-Hexyl analytical column measuring 100 × 2.1 mm ID × 2.6 μm. The methodology employed was derived from [38] utilizing the mobile phase of MS-grades water with 0.1% formic acid (A) and mass spectrometry (MS)-grades methanol with 0.1% formic acid (B), implemented via a gradient approach at a flow rate of 0.3 ml/min. The gradient commenced at 5% mobile phase B, progressively escalated to 90% over 16 minutes, maintained at 90% for 4 minutes, and thereafter reverted to their initial condition of 5% B by their 25-minute mark. The column temperature was sustained at 40°C, with an injection volume of 3 μl. The non-target screening was performed in a complete MS/dd-MS2 acquisition model with either positive or negative polarity or ionization state. The arbitrary units for nitrogen were 32, 8, and 4, and it served as a sheath, auxiliary, and purge gas. The auxiliary gas heater temperature was set to 30°C, the capillary temperature to 320°C, and the spray voltage to 3.30 kV. The resolution was 70,000 for full MS and 17,500 for dd-MS2 in both positive and negative ionization models, with a scanning range of 66.7 to 1000 m/z. The device was controlled using XCalibur 4.4 software from Thermo Scientific, Bremen, Germany. To ensure optimal instrument performance and robustness in mass accuracy investigations, their device was calibrated weekly in positive and negative ESI models utilizing Thermo Scientific Perce ESI join calibration solution (Waltham, MA).

Data analysis

All experiments were conducted in triplicate. Data are presented as mean values with a ± SD. Using the GraphPad prism and one-way analysis of variance (ANOVA), We identified significant results using a p-value of less than 0.05 using IBMSPSS Statistics 22 (ANOVA) software. All values are represented by (*).

RESULTS AND DISCUSSIONS

Antioxidant activity, TFC, and TPC against AS

The antioxidant efficacy of their extract and fraction was assessed utilizing the DPPH assay. Figure 1 illustrates the effectiveness of AS extract, produced with different solvents, in neutralizing DPPH free radicals compared to gallic acid across multiple concentrations ranging from 31.25 to 500 μg/ml. The DPPH scavenging activity of these different extracts is contingent upon concentration, augmenting with higher extract concentrations. The AS N-Hexane extract and fraction from each tested plant had the best antioxidant activity among the extracts analyzed.

Figure 1. Concentration-dependent DPPH inhibition activity of AS A. Extract of AS, B. Fraction of AS. [Click here to view]

Table 1 shows that the N-Hexane extract and fraction exhibit the best test parameter value compared to their antioxidant extracts and fractions, with the extract value (Antioxidant: 35.53 μg/ml, TFC: 14.96 mg QE/g, TPC: 25.61 mg GAE/g) and fraction values. (Antioxidant: 42.4 μg/ml, TFC: 15.51 mg QE/g, TPC: 25.31 mg GAE/g).

Table 1. The levels of antioxidant activity, TFC, and TPC against AS extract and fractions. [Click here to view]

Furthermore, we established a Pearson correlation among TPC, TFC, and the antioxidant activity of their test plants to ascertain the relationship between these biocompositions and their plants’ antioxidant activity (Table 2). A very positive association was identified between TFC and the antioxidant activity of the AS plant. The positive correlation suggests that the elevated antioxidant activity of the AS plant extract is attributable to a greater concentration of TPC. The AS fraction exhibits a favorable association and a robust link between antioxidant activity and TFC. Our findings corroborate earlier research indicating a strong correlation between TFC activity and antioxidant activity, as TFC is a prominent antioxidant agent in numerous medicinal plants.

Table 2. Pearson correlation between antioxidant activity, with fenolic content and flavonoid content. [Click here to view]

The research results show that the antioxidant test, TPC, and TFC in the n-hexane and ethyl acetate extracts of AS have good levels, with a very high Pearson correlation value between TFC and antioxidant activity, namely 0.900 for the extract and 0.898 for the fraction. This correlation indicates that flavonoid compounds significantly contribute to the antioxidant potential of AS, which is in line with various recent studies affirming the role of flavonoids as cellular protective agents with the ability to capture free radicals and reduce oxidative stress [39]. According to the latest journal, flavonoids not only function as antioxidants but also possess anti-inflammatory activity and can contribute to the prevention of degenerative diseases, such as cancer and heart disease. TPC, which includes other phenolic compounds, also enhances antioxidant activity, indicating synergy between flavonoids and phenolic compounds in the extract [39]. With its high levels of TFC and TPC, AS has the potential to be an effective natural source in traditional and modern medicine, supporting disease prevention strategies through increased intake of bioactive compounds [40]. Table 3 shows that AS extracts have potential as anti-cancer based on the IC50 values produced by N-Hexane and Ethyl acetate extracts have potential, with strong values shown by ethyl acetate extracts and followed by medium N hexane, and for methanol and ethanol extracts have no potential at all.

Table 3. IC50 value of AS extract with several different solvents. [Click here to view]

Cytotoxic activity based on MCF-7 cell viability

Cytotoxic assay results indicate a decline in their viability curves if MCF-7 cells beginning at five concentrations (31.25, 62.5, 125, 250, 500, and 1,000 μg/ml), which visibly diminished their growth curve. This interpretation of viability, based on three repetitions, is illustrated in Figure 3. N-hexane and ethyl acetate extracts demonstrated dases that inhibited 50% of MCF7 cell growth, ranging from 62.5 to 1,000 μg/ml. The IC50 values for each extract are calculated to ascertain the specific amounts that can inhibit the proliferation of MCF-7 cells, as illustrated in Table 4.

Table 4. IC50 value of AS fraction with several different fractions. [Click here to view]

Figure 2. Comparison of growth curves of all extracts of AS. [Click here to view]

Figure 3. The MCF-7 cell growth curve is compared to several AS fractions: (A) N-Hexane fraction of AS, (B) Ethyl acetate fraction of AS, (C) Butanol fraction of AS, and (D) Water fraction of AS. [Click here to view]

The growth curve in Figure 2 shows a variation in the growth curve between the solvents used. However, the AS growth chart with their ethyl acetate solvent decreased growth at a concentration close to their growth point of concentration of 62 μg/ml. Table 1 provides evidence of a 50% growth barrier concentration for their MCF-7 cell population. The IC50 calculation yielded a score of 62 μg/ml, indicating a patient inhibition of the growth of MCF-7 breast cancer cells.

The AS continued fractional testing with three repetitions. Inter-fraction variation results indicated a reduction in cell proliferation of their MCF-7 line corresponding to an increase in the concentration of the sample utilized (31.25, 62.5, 125, 250, 500, 1,000 μg/ml) (Figure 3). The N-hexane and ethyl acetate fractions exhibited potential anticancer IC50 values of 49 μg/ml (strong) and 89 μg/ml (strong), respectively (Table 2); the comparison of growth curves among AS fractions indicates a decline in growth at varying concentrations. Figure 4 shows the growth curve with variations between the fractions obtained. However, the growth graph of AS with N Hexane fraction decreased growth at a concentration close to the growth point concentration of 49 μg/ml. Table 4 provides evidence of a growth barrier concentration of 50% for the MCF-7 cell population. The IC50 calculation resulted in a score of 49 μg/ml, which indicates the inhibition of breast cancer cell growth against the MCF-7 cell line.

Figure 4. Comparison of growth curves of all fractions of AS. [Click here to view]

Table 4 shows the calculation of the IC50 value for each fraction, which is used to determine precisely what concentrations can inhibit the growth of MCF-7 cells.

A reduction in cell viability is noted at doses beginning at 5 μg/ml, with data indicating a diminishing growth curve if MCF-7 cells as their extract concentration escalates. The n-hexane and ethyl acetate extracts inhibit 50% MCF-7 cell proliferation at 62.5–1000 μg/ml. This indicates that these chemicals possess cytotoxic properties. The reduction in viability signifies that the active chemicals in their extract can compromise cell integrity and hinder the multiplication of cancer cells. These findings correspond with prior studies indicating that plant extracts can be anticancer agents, influencing apoptotic pathways and obstructing their cancer cell cycle [41]. Consequently, their subsequent stage involves further screening to isolate the bioactive compounds accountable for this anticancer activity and executing docking analysis to investigate the connections between these compounds and molecular targets. This discovery establishes a robust basis for advancing plant-derived cancer therapeutics, which may offer an alternative to traditional treatments that frequently include detrimental side effects.

Data variation among fractions, conducted with three repetitions, indicates a notable reduction in the proliferation of MCF-7 cells as the concentration of their sample escalates, particularly from 31.25 to 1,000 μg/ml. The reduction in cell viability signifies that their active chemicals in the n-hexane and ethyl acetate fractions effectively impede cancer cell proliferation. The IC₅₀ value for their n-hexane fraction is 49 μg/ml; their ethyl acetate fraction is 89 μg/ml, demonstrating significant anticancer activity in both fractions. These findings align with prior studies indicating that extracts from specific plants exhibit cytotoxic effects against various cancer cell types, including MCF-7, via mechanisms such as apoptosis induction and cell cycle inhibition [42,43]. This discovery underscores the necessity of further investigating the bioactive chemicals in that fraction, which may be developed as novel therapeutic agents for cancer treatment.

Hexane extract and fractions tend to extract lipophilic compounds such as specific terpenoids and flavonoids known to have anticancer activity. These compounds can interact with the cancer cell membrane, disrupt the cell proliferation process, and trigger apoptosis (programmed cell death). On the other hand, although polar and capable of extracting polar compounds, ethanol, and methanol extracts may not contain high concentrations of bioactive compounds that effectively inhibit the growth of MCF-7 cells. Previous research by Zhang et al. [44] showed that lipophilic compounds have a better affinity for targeting signaling pathways involved in cancer growth [45]. This may explain why ethanol and methanol-based extracts do not exhibit the same potential. Butanol solvent on extract and fraction with medium polarity may also not contain the optimal active compounds to inhibit cancer cell growth. According to Chehelgerdi [46] bioactive compounds isolated from medicinal plants often have varying efficiencies depending on the type of solvent used. In this case, the components extracted with butanol may not be sufficiently effective in targeting the active oncogenic pathways in MCF-7 cells [47].

Analysis of AS N-Hexane fraction compounds with LC-HRMS

This study uses an LC-HRMS analysis to identify the sensitivities present in the AS N-Hexane fraction. LC-HRMS enables comprehensive molecular fingerprinting in a sample, following up on the procedure of Windarsih et al. [38]. The analysis's results indicate the predicted relationship between the identified subject and the m/z cloud base data. A high-resolution spectrometer analyses various molecules using the MS and MS/MS spectra presented in the mzCloud basis data. This spectrum can be used as a reference when looking up uncommon words. Identification of molecules is based on high-resolution data from single molecules and all fragments of molecules. The Compound Discover tool compares the fragmentation patterns and molecular ions with those found in the m/zCloud database to determine the best match. In the case of a compound match, the software will show ranges from 0 to 100, with a higher score indicating a more significant number of the best matches of the number match of ions [38].

A total of 4 compounds were successfully filtered by considering their best value to be from cloud best match confidence, with a minimum value ranging from 95% to 100%, to proceed with silicon analysis and obtain screening results in the form of potential compounds that can reduce cancer cell proliferation. Screening Results are shown in Table 5.

Table 5. Prediction of phytochemical compounds Fraction of ethyl acetate AS. [Click here to view]

Information regarding the retention time (RT) on the total ion current (TIC) graph in phytochemical screening results using LC-HRMS is essential for identifying and analyzing compounds. Each compound has a characteristic RT, so the peaks that appear on the TIC graph can be used to determine when a compound elutes from the chromatography column, as shown in Figure 5.

Figure 5. TIC graph in phytochemical screening results (Red circle: α-Linolenic acid; Blue Circle: Galaxolidone; Yellow Circle: Pinocembrin; Purple Circle: Afzelin). [Click here to view]

Table 6. Chemical formula and MS-MS spectra. [Click here to view]

The LC HRMS testing results indicated that four substances were accurately identified and selected based on optimal mzCloud match values, with scores between 95% and 100%. This filtering procedure is essential for their methods, such as silicon analysis, to identify possible chemicals limiting cancer cell proliferation. These characteristics derived from existing, including Measured (m/z), Exact mass m/z, Cloud Best Match confidence (%), and Product coins (m/z), are essential for their identification of pertinent substances. The measured m/z offers insights into the mass-to-charge ratio, whereas the exact mass m/z facilitates precise compound identification [48]. The Cloud Best Match confidence (%) signifies the degree of assurance concerning the data match, guaranteeing that the chosen compounds are representative and pertinent. Production (m/z) data elucidate the fragmentation of compounds, adding to the comprehension of their chemical structure and potential biological action. By synthesizing the outcomes from LC HRMS and in silico analysis, researchers may effectively forecast interactions between drugs and molecular targets, thereby expediting the development of anticancer medicines. Our research findings align with prior studies demonstrating that a multidisciplinary approach in drug development, incorporating LC HRMS technology and in silico analysis, can improve the efficacy and safety of cancer therapies derived from natural compounds [49,50]

The LC-HRMS test found a group of phytochemicals that are very interesting. These are Galaxolidone, α-linolenic acid, pinocembrin, and afzelin. The m/z values indicate their easy identification. Galaxolidone (279.23 m/z) and α-linolenic acid (272.18 m/z) were detected with perfect Cloud Best Match. Confidence values, 100% and 93.1%, respectively, indicate that these compounds have strong potential to provide biological effects. Also, pinocembrin (257.08 m/z) and afzelin (431.09 m/z) have high confidence values (95.9%), which means that these compounds may help the extract work as expected in the pharmaceutical field.

Galaxolidone is a phytochemical compound synonymous with estradiol and is an aromatic compound that can reduce inflammation and protect cells from damage. It can stop cancer cells from multiplying by controlling signaling pathways in the cell cycle and induced apoptosis mechanism [51]. Galaxolidone or Galaxolide is a sesquiterpene lactone, a chemical frequently occurring in flora. It is recognized for its possible anti-inflammatory and antibacterial characteristics [52]. Galaxolidone has been extracted from multiple plant species, including those within their Asteraceae family. Flavonoids are recognized for their antioxidant characteristics and possible health advantages [53]. Pinocembrin has been investigated for its prospective anti-inflammatory, neuroprotective, and anticancer properties [54]. α-Linolenic acid, an omega-3 fatty acid, is also known to help fight cancer. Studies have shown that this compound can cause apoptosis in MCF-7 cells and stop cancer cells from spreading and migrating. Various plants contain pinocembrin, a flavonoid that exhibits cytotoxic effects against MCF-7 cells and can inhibit signaling pathways in cancer cell growth [55].

Afzelin, a flavonoid glycoside, possesses significant potential in inhibiting the growth of MCF-7 cells. Previous research has shown that afzelin can induce cell death through apoptosis and disrupt the proliferation process of cancer cells [56,57]. Overall, these compounds show potential as promising anticancer agents, and further research is needed to understand their mechanisms of action and effectiveness in cancer therapy. Because there is much evidence that these compounds can fight cancer, AS can be seen as a valuable natural source for making cancer treatments that work better and are safer.

CONCLUSION

With a value of 35.53 μg/ml, total phenol is 25.61mg GAE/g, and total flavonoid is 29.50 mg QE/g, this study shows that N-hexane extract may be a better antioxidant than other test materials. This is true for both the extract and fraction of AS. These data are also supported by the inhibition of MCF-7 cell growth with an IC50 value of 42 μg/ml in the n-hexane extract, which is the best IC50 value. This is a further indicator for evaluating the LC-HRMS phytochemical profile of N-Hexane AS extract, and four phytochemical compounds are obtained. Galaxolidone, linolenic acid, pinocembrin, and afzelin. 

ACKNOWLEDGMENTS

This work was funded by Research Grant 2024 from the Health Research Organization, National Research and Innovation Agency (BRIN), Indonesia. We also thank KS Gunung Kidul (Umar Anggara Jenie) Kawasan Sains Umar Anggara Jenie BRIN Yogyakarta for the LC-HRMS analyses.

AUTHOR CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.

CONFLICTS OF INTEREST

The authors report no financial or any other conflicts of interest in this work.

ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.

DATA AVAILABILITY

All data generated and analyzed are included in this research article.

PUBLISHER’S NOTE

All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY

The authors declares that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.

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