Total Flavonoid Content, Antioxidant Activity, and Cytotoxicity Profiling of Astragalus spruneri Extracts using IncuCyte® Live Cell Analysis

1. Introduction

Cancer represents one of the most prevalent causes of death worldwide, comprising about 10 million globally in 2020 [1]. Notably, breast cancer is the most commonly diagnosed malignancy across countries, with about 2.3 million new cases, making up 11.7% of all cancer diagnoses. According to estimates of the World Health Organization [2], the incidence in developed nations was 88% higher, whereas mortality rate was 17% lower compared to developing regions. In contrast with other regions, the significant incidence increases in Europe and Oceania were preferentially detected in estrogen receptor-positive cancer, while estrogen receptor-negative cancer rates are falling. Overall, it continues to be the highest mortality among women with a significant public health burden [3].

Investigations spanning in vitro assays, animal models, and clinical trials have largely attributed natural products as necessary candidates for preclinical development and future therapeutic interventions. To this purpose, numerous plant extracts and bioactive compounds in part revealed to promote antitumor effects [4,5,6,7,8,9,10]. Interestingly, plants or vitamins were selected by 60% of patients as their most favoured form of cancer therapy [11]. Furthermore, the US National Cancer Institute (NCI) from the 1960s to 1980s tested 114,000 natural product extracts and compounds for their potential antitumor activity [12].

A hallmark in carcinogenesis, the disruption of the balance between cell proliferation and apoptosis, may be counteracted by plant-derived biomolecules. Phytochemicals regulate inappropriate molecular activity underlying processes, leading to a curative approach [13]. Subtle alterations in cell death can lead to cell loss and remain a central focus of biological research, bringing to light the importance of targeting these pathways. In this study, cell death is analyzed using Incucyte® Cytotox NIR dye (Sartorius 4646), enabling real-time, robust, and precise measurements. Unlike endpoint assays, which provide only a single snapshot of cell state and exclude time-dependent information, real-time kinetic assays capture the entire time-course of the cell death response. This continuous monitoring reduces inter-sample variation between replicates and minimizes susceptibility to human error, thereby improving precision in longitudinal assessment. The Incucyte® Cytotoxicity Assay enables real-time monitoring of cell death kinetics by automatically scrutinizing dying cells over time using Incucyte® Cytotox NIR Dye. This dye remains non-fluorescent in viable cells but, upon loss of membrane integrity, it binds nucleic acid and emits a near-infrared fluorescent signal at 685-756 nm.

The genus Astragalus is among the largest vascular plant genera of herbs and small shrubs, within the family Fabaceae, distributed across the temperate regions of Europe, Asia, and North America, with particularly pronounced diversity in the Mediterranean and Central Asia. Species of Astragalus have been widely explored for their rich content of bioactive metabolites, including flavonoids, saponins, polysaccharides, and phenolic acids, many of which have demonstrated cytotoxic and antiproliferative activities both in vitro and in vivo [14,15,16,17]. Comprehensively characterized species, such as A. membranaceus, mongholicus etc, are already recognized in both traditional medicine and modern pharmacology for their anticancer potential [18,19,20,21,22], thereby highlighting the genus as a valuable reservoir of antitumor plants.

Nonetheless, Astragalus spruneri Boiss., a perennial, prostrate, ground-hugging herbaceous plant endemic to Southeast Europe and Turkey, remains insufficiently explored to date. It has a limited distribution in its natural habitat and has not been specified for medicinal purposes in traditional medicine. In early studies, phytochemical profiling of Astragalus spruneri demonstrated a valuable reservoir of flavonoids and saponins in the defatted extract, which collectively delineate the plant’s potent antioxidant signature [23,24]. The total volatile compounds of A. spruneri, inhibited REH cells (human acute lymphoid blast leukaemia cells) in a concentration-dependent manner, although their effect was moderate relative to that of other Astragalus species [25]. Therefore, further research with an emphasis on its cytotoxic profile is required to clarify whether it can feasibly contribute to anticancer activity.

Herein, a well-established approach was used to evaluate variation in total phenolic content, total flavonoid content, and antioxidant potential of extracts obtained from aerial parts of Astragalus spruneri using solvents with different polarities to enable the differential extraction of bioactive constituents, including phenolic and flavonoid compounds. These extracts were subsequently investigated using label-free live-cell analysis for their ability to selectively induce cell death in MCF-7 cells, to gain new insights into the tissue-specific and solvent-dependent bioactivity of A. spruneri. In addition, morphological changes and PUMA protein expression were assessed to distinguish further insights into the cellular mechanisms associated with extract-induced cell death. Collectively, this study provides the first report describing kinetic cytotoxic responses of A. spruneri using real-time live-cell analysis to enlighten the understanding of this underexplored species as a potential source of bioactive compounds with anticancer properties.

2. Materials and Methods

2.1. Plant Material

Astragalus spruneri (Fabaceae) was collected from regions with temperate and arid climates in Funare Village, Elbasan, Albania (729 m, 41°16′01″N, 20°17′27″E0), Albania (2025), and authenticated by a botanist at the Department of Biology, University of Elbasan “A. Xhuvani”, Albania. A voucher specimen was deposited at the departmental herbarium for future reference. This plant was shade-dried and then ground.

2.2. Preparation and Extraction Process

Ten grams of dried powdered (flower, leaf, and stem) Astragalus spruneri was successively extracted by dynamic maceration with 100 ml of solvents and water at room temperature for 24 hours (3 times each solvent). The extraction was carried out using ultrasound-assisted extraction (UAE) for 60 minutes at 35–40 °C. The recovery of phenolic compounds from different parts of the A. spuneri was performed consecutively by solid-liquid extraction methods using two different solvent systems: methanol: water (60:40) and ethanol: water (80:20). The phenolic extract was then filtered using a 0.45 µm membrane and stored at -20°C until further analysis.

2.3. Determination of Total Phenolic Content (TPC)

The total phenolic content (TPC) was determined using the Folin–Ciocalteu method, as previously described [26]. Polyphenols were quantified based on the prepared calibration curve (0–10 mgGA/mL; y=1.3839x -0.0006, where R2=0.9997) using standard solutions of gallic acid obtained from Sigma-Aldrich.

The assay sample, 0.5 mL of phenolic extract, 10 mL of distilled water, and 2.5 mL of Folin-Ciocalteu reagent were added to each test tube. The mixture was vortexed and incubated in the dark for 10 minutes. Afterwards, 2 mL of 7.5% sodium carbonate was added to each tube and mixed again. All samples were incubated in a water bath previously preheated to 40°C and allowed to react for 40 minutes. Absorbance was measured at 760 nm at room temperature using a UV-Vis spectrophotometer. The results were expressed as mg gallic acid equivalent per gram of dried plant (mgGAE/g). All measurements were performed in 3 replicates.

2.4. Determination of Total Flavonoid Content (TFC)

The total flavonoid content (TFC) of extracts from different parts of Astragalus spruneri was determined using the aluminum chloride colorimetric method described by [27], with minor modifications. A calibration curve was prepared using standard quercetin (100-1000 μg/mL). Briefly, 1 mL of the dissolved extract was mixed with 4 mL of distilled water in a test tube. Then, 0.3 mL of a 5% sodium nitrite (NaNO₂) solution was added, and the mixture was allowed to stand for 6 minutes. Subsequently, 1 mL of 10% aluminum chloride (AlCl₃) solution was introduced, and the reaction mixture was left to stand for another 6 minutes. Finally, 0.4 mL of distilled water and 4 mL of 4% sodium hydroxide (NaOH) solution were added, resulting in a colour change from yellow to red. The mixture was then allowed to react in the dark for 15 minutes, after which the absorbance was recorded at 510 nm. The total flavonoid content was expressed in milligrams of quercetin equivalents per gram of oven-dried plant powder (mg QE/g OP).

2.5. DPPH Free Radical Scavenging Activity Assay

dical scavenging activity of Astragalus spruneri extracts was assessed using the stable 1,1-diphenyl-2 picrylhydrazyl (DPPH)-free radical activity [28]. In each test tube, 40 μL of the extract was stirred with 1960 μL of methanol and 2000 μL of 0.1 mM DPPH solution. The blend was then shaken at room temperature and left to react in a dark place for 40 min. The absorbance of each solution was determined at 515 nm using a spectrophotometer, with methanol as a blank. The control consisted of 0.1mM DPPH in methanol. The equation for the total antioxidant activity was employed to establish the relationship as a percentage of total inhibition of DPPH, plotting the following formula:

% DPPH$={\displaystyle \frac{Ao-Ac}{Ao}}x100$.

Where: A₀ was the absorbance reading of the DPPH control measured at the initial time, whereas Ac represents the absorbance reading of the extract after a 40-minute rest in a dark place.

2.6. MCF-7 Cell Culture

MFC- 7 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS). Cells were seeded in 6-well tissue culture plates (TPP) and incubated in a humidified incubator at 37°C with a gas mixture of 5% CO₂ and 20% O₂. The medium was refreshed every 2-3 days to maintain appropriate growth conditions. For passaging, cells were detached using TrypLE Express (Gibco) and subcultured at a 1:6 split ratio.

2.7. MFC-7 Cytotoxicity Screening

MFC-7 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and treated with the target compound at concentrations of 50, 100, 200, and 400 µg/mL, prepared from a 1:100 stock solution in DMSO. Corresponding controls were included: 1% DMSO (vehicle control) and untreated cells. During the treatment period, the medium was supplemented with Incucyte® Cytotox NIR dye (Sartorius, catalogue no. 4646) at a final dilution of 1:1000. Cell imaging was performed every 2 hours using a 4x objective with a 400 ms exposure time on the Incucyte® SX5 Live-Cell Analysis System (Sartorius). Changes in fluorescent intensity over the time course were quantified by calculating the total integrated intensity based on NIR fluorescence using the Incucyte software. Representative bright-field images were processed using Fiji (ImageJ, NIH, Bethesda, MD, USA) to enhance clarity for visualisation. Identical adjustment parameters were applied to all images.

2.8. Western Blot Analysis

Cell lysates were prepared from pelleted MCF-7 cells by adding cold RIPA Lysis Buffer (MILLIPORE, 20-188) with protease inhibitor mix (EDTA-free, Roche), followed by a 30-minute incubation on ice and pipetting every 10 minutes. Supernatants were retained after 5-minute centrifugation (4°C) for SDS-PAGE. 20 µg of protein from the cell lysates were mixed with NuPAGE LDS Sample Buffer (Thermo Scientific, NP0007) and NuPAGE Sample Reducing Agent (Thermo Scientific, NP0004) followed by an incubation at 70°C for 10 minutes. Samples were separated on NuPage 4-12% Bis-Tris Gel (Thermo Scientific, NW04120) in Novex NuPAGE MES SDS Running Buffer (Thermo Scientific, NP0002) alongside PageRuler Plus Prestained Protein Ladder (Thermo Scientific, 26619). The proteins were transferred to a PVDF membrane by iBlot2 Life Technologies Apparatus. PUMA antibody (Proteintech, 55120-1-AP, dilution 1:500) followed by GAPDH (Santa Cruz Biotechnology, Sc-47724, 1:10 000) was incubated with the membrane overnight at 4°C. Secondary peroxidase-conjugated anti-rabbit or anti-mouse IgG (Dako) incubation was performed at room temperature for 1 hour. The signal was obtained using the Thermo Scientific Super Signal West Pico Plus Chemiluminescent substrate kit and detected by an Amersham Imager 600.

2.9. Statistical Analysis

All measurements for bioactive compound, antioxidant activity, and PUMA were conducted in triplicate. Data were expressed as the mean ± SD (standard deviation). P values less than 0.05 were considered statistically significant. The Pearson correlation coefficient test was performed on values obtained for TPC, TFC, and DPPH. Nonlinear regression was carried out to estimate the corresponding IC50 values. Data were analyzed using GraphPad Prism version 11.0.2 for Windows (GraphPad Software, Boston, Massachusetts, USA). One-way and two-way ANOVA followed by Dunnett's or Tukey's post hoc test were used to compare differences among distinct groups.

3. Results and Discussions

3.1. Total Flavonoid, Total Phenol Content, and Antioxidant Capacity Determined by the DPPH Assay

Phenolic and flavonoid compounds have been widely recognized for their diverse biological activities and are considered key phytochemicals responsible for many of the health-promoting properties of medicinal plants. These secondary plant metabolites are involved in normal growth and development while serving as important components in defence mechanisms against oxidative stress and reactive oxygen species (ROS).

Total phenolic content, flavonoid content, and DPPH Free Radical Scavenging activity are presented in Table 1 and Table 2. Overall, the accumulation of these bioactive compounds varied considerably among the tested extracts, depending on the extraction solvent and plant organ. The analysis revealed that the total flavonoid content (TFC) differed between 5.59±0.18 and 10.29±0.81 mg QE/g extract in the methanol extract (Table 1) and from 8.03±0.67 to 13.31±1.06 mg QE/g extract in the ethanol extract (Table 2). On the other hand, total phenolic content (TPC) was higher in the methanolic extracts (6.94±0.56 to 11.49±1.31 mg GAE/g) than in the ethanolic extracts (4.58±0.42 to 8.97±0.35 mg GAE/g). The greatest levels of phenolics and flavonoids were detected in the flower methanol extract (11.49±1.31 mg GAE/g and 10.29±0.81 mg QE/g) and the ethanol extract (8.97± 0.35 mg GAE/g and 13.31±1.06 mg QE/g), correspondingly.

The TFC observed in previously studied samples showed substantial variance depending on the Astragalus species and the plant part analyzed. Platikanov [29], reported a notably high flavonoid concentration in the aerial parts, reaching 49.13 ± 0.51 mg REs/g extract, whereas the highest phenolic content was observed in the leaves with the value of 26.34 ± 0.50 mg GAEs/g extract. Moreover, leaves of A. glycyphyllos and A. cicer [30] contained higher flavonoid levels compared to the stem extracts. This pattern aligns with the findings of the present study on ethanol extract.

A similar distribution pattern of TPC and TFC was reported by Arumugam [16], with generally lower values in plant parts of A. glycyphyllos. However, the flowering plant investigated in our study accumulated higher levels of total flavonoid content in flowers and leaves than those reported for A. cicer, although these levels were lower than those observed in A. glycyphyllos and A. ponticus [16]. In addition, the methanolic extracts of leaf and flower displayed the highest DPPH free radical scavenging activity at 95.80±0.1%, highlighting stronger DPPH radical-scavenging activity than the stem (Table 1) extracts. Conversely, ethanolic extracts underscore no variability in antioxidant capacity among different plant parts (Table 2).

3.2. In Vitro Cytotoxicity IncuCyte® Live-Cell Assay of A. spruneri Extracts in MCF-7 Cells

Cell death represents a critical parameter for evaluating the potential bioactivity of plant extracts. To assess the ability of Astragalus spruneri (AS) to induce cell death, different extracts were tested for in vitro cytotoxicity against estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) human breast adenocarcinoma cell line MCF-7. Congruently, we evidenced fluorescent signal accumulation and proceeded with visible morphological changes in cells using bright-field microscopy to compare the relative effects of different AS extracts.

Hence, straightforward data analysis of real-time monitoring of NIR signal dynamics was performed with the IncuCyte® Live-Cell Analysis System, enabling the assessment of cellular responses over time while minimizing human intervention. This approach reduces variability associated with endpoint measurements and improves the precision and reproducibility of NIR signal quantification across time points.

The influence of stem extracts of Astragalus spruneri (ASS) on cell death and cell morphology was initially evidenced using IncuCyte® Cytotox NIR dye, which penetrates cells following membrane compromise, binds to nuclear DNA, and emits a fluorescent signal. To this end, four different concentrations of ASS (50, 100, 200, and 400 µg/mL) were applied to MCF-7 cells for 72 hours. As demonstrated in Figure 1A,B, the accumulation of the NIR-positive cells at 50 or 100 µg/mL did not differ appreciably from that observed in untreated cells or those treated with 1% DMSO. Cytotoxicity became evident at 200 µg/mL and more prominent at 400 µg/mL, where the NIR signal (Figure 2A) was significantly elevated by approximately 2-fold after 72 hours compared with 48 hours of incubation, and more than 4-fold compared with 24 hours. The data indicate that the ethanolic extract induced cytotoxic effects only at concentrations ≥200 µg/mL. As the treatment dose increased, cellular damage accumulated over time, leading to progressively greater cytotoxic effects.

To further highlight the distinct effects of ASS ethanolic and methanolic extracts, we analyzed the cumulative maximal NIR signal. Notably, incubation of MCF-7cell lines with ethanolic ASS extracts (Figure 2B) resulted in 7.6-fold greater fluorescence intensity over time at 400 µg/mL compared with 200 µg/mL. Furthermore, total maximum NIR intensity was markedly altered, showing a 5.9-fold increase in cells treated with 400µg/mL ethanolic ASS extract compared with those exposed to ASS methanolic extracts at the same concentration. Thus, treatment of MCF-7 cells with higher concentrations of ethanolic extracts led to progressive disruption of the plasma membrane and a corresponding increase in NIR dye accumulation.

Furthermore, the in vitro cytotoxic activity of crude ethanol and methanol leaf extracts of A. spruneri (ASL) was characterized against the MCF-7 cell line after 24, 48, and 72 hours of exposure to varying concentrations relative to the vehicle control. Figure 3A‒C demonstrates a dose-dependent enhancement of cytotoxicity induced by treatment. At 200 µg/mL, the ethanolic crude extract increased the maximum NIR signal by approximately 73% after 24 h, 90% after 48 h, and 60% after 72 h (p < 0.001) compared to 1% DMSO. Alternatively, methanolic crude extract induced comparatively modest changes, reaching optimal accumulation of approximately 15% at 24 h, followed by a reduced activity of about 10% persisting through 48 h and 72 h.

Treatment with 50 µg/mL of the ethanolic extracts (Figure 3D) exerted only negligible cytotoxic effects. Moreover, concentrations above 100 µg/mL induced prominent membrane damage, as evidenced by an increase in Cytotoxic NIR signal, which rose nearly 6.4-fold at 200 µg/mL and 8.5-fold at 400 µg/mL compared to the vehicle control. The results of the ethanolic crude extracts appear to reach their peak cytotoxic potential at maximal concentration after the first 24 h of treatment (Figure 3E,F), while prolonged exposure produced no additional increases in treatment-induced cytotoxicity. At concentrations of 200 µg/mL and 400 µg/mL, ethanol extract of the leaf showed significant maximum NIR signals of 73% and 95% over 24 h, respectively, compared to the vehicle control. Meanwhile, the methanol leaf extract produced responses of 15% and 35%, respectively, under the same conditions. Thus, the most striking cytotoxic effect occurred within the first 24 hours, and the ethanol leaf extract was consistently more potent than the methanol extract throughout the treatment period. In addition, exposure to the methanolic extract produced only a noticeably weaker and divergent effect.

Cytotoxicity was quantified kinetically, and the overall response was summarized as the area under the fluorescence curve (AUC) (Figure 4A). The AUC showed a concentration-dependent increase in cumulative Cytotox NIR signal over the measurement period. The effect became notable at 100 µg/mL and reached its greatest effect at 400 µg/mL, resulting in a 1.74-fold increase compared with 200 µg/mL ethanol extract treatment. On the other hand, methanol extracts of ASL elicited considerably lower cumulative cytotoxic responses in a dose-dependent pattern.

Based on the normalized dose–response curves, the half-maximal effective concentrations (EC50) of the tested ASL extracts were calculated by nonlinear regression using a four-parametric logistic model [log(concentration) versus response variable slope]. The EC50 values and their 95% confidence intervals were calculated from normalized and transformed Cytotox NIR responses (Figure 5). The estimated EC50 results for ethanolic extracts were 151.5, 125, and 158.1 μg/mL over 24, 48, and 72 h, respectively. Breast cancer MCF-7 cells showed the highest sensitivity towards the ASL ethanol extract, with an EC50 of 125 μg/mL determined after 48 h of treatment, compared to the methanol extract EC50 >200 μg/mL.

The cytotoxic activity observed for ASL extracts differed from previous findings reported on other Astragalus species [31], which documented IC50 values ranging from 601.00 to 1307.00 μg /mL for ethanolic extracts of Astragalus globosus and Astragalus breviflorus, respectively, against the MCF-7 cell line using the MTT assay. Among other Astragalus species that have been investigated for cytotoxic activities in previous studies, Astragalus polysaccharides [32] showed an antiproliferative effect against the MCF-7 cell line, with an IC50 of 945 µg/ml at 24 h. Although direct comparison with the EC50 value obtained in the present study is constrained by differences in assay endpoints and data analysis methods, the ASL ethanolic extracts nevertheless exhibited moderate cytotoxic activity under kinetic Cytotox NIR assay conditions used in this study.

3.3. Morphological Alteration in MCF-7 Cells Induced by Astragalus spruneri Leaf Extracts

The cytotoxic performance of the Astragalus spruneri leaf extracts against human breast malignancies (MCF-7) demonstrated marked variation based on both the extraction solvent and applied concentration. The bright-field micrograph of MCF-7 cells treated with ethanolic extracts of AS leaf revealed a prominent dose- and time-dependent progression (Figure 5). Following 4 hours of treatment at 100 ug/ml, MCF-7 cells predominantly retained their baseline features, exhibiting active growth, polygonal morphology, and tight intercellular connections, while morphological alterations developed more gradually. In stark contrast, the highest concentration of 400 ug/ml elicited an immediate response, evident as early as 4 hours, with visible, rounded, disrupted cells and a pronounced reduction in confluence.

Prolonged incubation and higher concentrations triggered a dramatic morphological shift. The dense monolayer starts to disintegrate over 12 and 24 hours, giving way to distinct structural features characteristic of apoptosis, including significant cell shrinkage, rounding, loss of substrate anchoring, and cellular fragmentation into spherical apoptotic bodies. The primary pathway of cell death appeared to be apoptotic-like changes; the extensive cell degradation and severe monolayer retraction observed by 24 hours suggest that the cells ultimately progress to secondary necrosis in the late stages of treatment. ASE treatment led to a sustained and significant increase in NIR signal with an earlier onset of cytotoxicity at 15 min of exposure (Figure 3F), and was also evident after 60 min and 105 min of exposure to MCF-7 cells. Bright-field microscopy revealed the onset of cell damage starting from 4 h of incubation at 400 µg/ml, 200 µg/ml, and 8 h at 100 µg/ml (Figure 5).

We simultaneously evaluated both the ethanolic and methanolic stem extracts of AS. However, because these samples showed only minor Cytotoxic NIR response under all tested conditions, their data did not warrant further kinetic or morphological characterization. Treatment with the methanolic extract of the leaf (Figure 6) led to alterations in cellular and nuclear morphology, indicative of cytotoxic activity. At 100 ug/ml, the extract did not exert a detectable cytotoxic effect on the cell line throughout the 72-h timeline. Instead, cells preserved their standard architecture, characterized by tightly cohesive, proliferating epithelial-like clusters, and promoted cell growth.

At 200 ug/ml, changes in cellular shape were first observed in a subset of MCF-7 cells after 6 hours of exposure, whereas by 24 h, the majority of cells exhibited evident changes characterized by cell rounding and condensation. The response was considerably more prominent at 400 ug/m. At this treatment, changes in cell morphology were already apparent in the majority of cells after 6 h, while by 24 h, virtually all cells had undergone marked morphological deterioration accompanied by accumulation of cellular remnants.

3.4. Association between Phytochemical Composition and Cytotoxic Activity

The previous finding of the higher DPPH radical-scavenging activity observed in methanolic leaf extracts was associated with a strong correlation between flavonoid content (R=0.975) and phenolic content (R=0.691) (Table 1), suggesting that these compounds may contribute substantially to antioxidant properties. Although, the extract’s biological activity may be explained by either a single active phytochemical or the synergistic interaction of multiple phytochemicals. The anticancer effect of ASL may primarily reflect the contribution of flavonoids as a major phytochemical profile. These compounds can disrupt redox homeostasis by increasing intracellular ROS levels, which in turn promotes oxidative stress and cell death [33].

The significantly higher leaf flavonoid content compared with the stem extracts was accompanied by greater cytotoxic activity against MCF-7 cells. Moreover, phenolics and flavonoids exhibited a moderate negative correlation with DPPH radical scavenging activity (R = ‒0.53). Simply, this association supports the hypothesis that flavonoids may promote cell death pathways in MCF-7 cells. Previous research has revealed 12 major flavonoids in Astragalus spruneri and three rare flavonoids from the aerial part of the plant [23,34], which may participate in triggering the obvious cytotoxicity. It is known that isolated flavonoids from Astragalus species have demonstrated antiproliferative and cytotoxic effects in different cancer models, such as human leukemia cells (HL-60), hepatocarcinoma (SMM7721 and HepG2), and breast cancer cells [35,36,37,38]. Total flavonoids extracted from the roots of Astragalus membranaceus (IC50=98.63 ug/ml), as well as its isolated compound calycosin, effectively inhibited the proliferation of K562 [9]. The use of solvents with varying polarities enables the differential extraction of bioactive constituents including phenolic compounds, flavonoids, and other secondary metabolites from A. spruneri. Even so, the potential contributions of other phytochemicals such as saponins identified in Astragalus spruneri [23,34] should not be overlooked, as they are reported to possess potent cytotoxic activity in Astragalus species [35].

3.5. Association of PUMA with MCF-7 Cells

Subsequently, we explored the involvement of the pro-apoptotic regulator PUMA by Western blot analysis. The PUMA level was normalized to GAPDH and compared across ethanol and methanol in leaf and stem extracts, as well as vehicle control (DMSO 1%) and untreated cells (Figure 7). PUMA expression was evaluated at 6 h, and the reduced protein abundance may suggest transient regulation of PUMA expression or degradation during progression of extract-induced cell death. Notably, the extract producing the strongest cytotoxic effect exhibited the lowest detectable PUMA protein level. Since the Incucyte data showed that the ethanolic extract (stem, leaf) induced greater cytotoxicity, more cells likely progressed to later stages of cell death than those treated with the methanolic extract (stem, leaf). The inverse relationship was stronger for the leaf extracts, while stem extracts induced a weaker cytotoxic response and smaller changes in PUMA expression.

PUMA plays a critical role in mediating cell death pathways [39,40], facilitating crosstalk determined by the severity of extracellular or intracellular stress-induced stimuli. In MCF-7 cells, estrogen inactivates the p53 pathway, downregulating PUMA transcription through binding upstream of its transcription start site [41,42]. Instead, our results indicate that the greater cytotoxic activity of the ethanolic extracts may be associated with more rapid progression of regulated cell death and temporal regulation of PUMA expression. Thus, further investigations are required to characterize the molecular mechanisms and determine the type of regulated cell death induced by A. spruneri extracts in MCF-7 cells, as well as the role of PUMA in this process.

4. Conclusions

The Cytotox NIR assay provided new insights into distinct extract- and tissue-dependent cytotoxic responses of Astragalus spruneri extracts against MCF-7 cancer cells, with ethanolic leaf extracts exhibiting the most prominent effect. These findings provide the first comprehensive kinetic characterization of the cytotoxic activity of AS, suggesting that flavonoid-enriched ethanolic leaf extracts induced an earlier onset and more sustained cytotoxic response. The results support A. spruneri as a promising source of bioactive compounds and highlight the importance of integrating real-time cellular analysis for evaluating its biological potential. Further investigation is required to elucidate the effects of Astragalus spruneri ethanolic leaf extract on cell death, the underlying molecular pathways, and the active constituents responsible for its antitumor potential.