Assessment of cytotoxic, anti-proliferative, anti-metastatic, and morphometric effects of Tecoma stans extracts against MDA-MB-231 human breast cancer cell line

Abstract

Background

Breast cancer remains the most commonly diagnosed cancer type among women, with a high mortality rate due to metastasis. Chemotherapy remains very aggressive, with burdensome side effects on the body. Tecoma stans is an ornamental plant widely used to treat many diseases according to its various pharmacological properties. This study aimed to investigate the cytotoxic, antiproliferative, and antimetastatic activities of Tecoma stans leaves & flowers methanolic extract (TSLME &TSFME) on a highly metastatic MDA-MB-231 breast cancer cell line.

Methods

Gas chromatography-mass spectrometry (GC–MS) analysis of TSLME and TSFME was carried out to identify present bioactive compounds. TSLME and TSFME were used to assess their cytotoxic (Trypan Blue Exclusion Assay), antiproliferative (MTT Assay), and anti-metastatic activity (Wound Healing Assay) on the triple-negative breast cancer cell line MDA-MB-231, along with their impact on the morphology of metastatic cells.

Results

TSLME showed significantly higher cytotoxicity on MDA-MB-231 cancer cells compared to TSFME at 800 µg/mL. On the contrary, at 200 and 100 µg/mL, TSFME exhibited a more robust antiproliferative effect than TSLME. Additionally, TSLME and TSFME exhibited a significant inhibitory effect on the cell motility upon 24 h at 48 h incubations. Moreover, TSLME and TSFME induced apoptosis in MDA-MB-231 cells and altered their morphology at different concentrations (Decrease in the cell surface area, plasma membrane bleb formation, and nuclear condensation). TSLME and TSFME significantly reduced the cell body and field diameter and process thickness of MDA-MB-231 cells at 800 µg/mL (TSLME > TSFME) and at 200 µg/mL (TSFME > TSLME) which can be an indicator of cell cycle arrest as a response to cellular stress or DNA damage. The pharmacological activities demonstrated in this research by TSLME and TSFME can be attributed to numerous bioactive molecules detected through GC–MS analysis with anticancer activities. These compounds may have acted singularly or synergistically in a concentration-dependent manner.

Conclusion

This study revealed the cytotoxic, antiproliferative, and antimetastatic effects of Tecoma stans. Following in-depth in vivo research and clinical trials, Tecoma stans could therefore be used as a promising chemotherapeutic drug against highly metastatic human breast cancer cell lines.

Background

Cancer is defined as the continuous, unregulated proliferation of body cells [1]. The loss of growth control mechanism in the cell cycle exhibited by cancer cells is a result of accumulation of mutations in DNA, leading to alterations in the cell behavior that distinguish cancer cells from their normal counterparts [2]. This disorganized nature of cancer cells is due to genetic factors such as accumulation of mutations, or epigenetic dysfunction of key genes (oncogenes), which play an important role in cell division and cell death control by apoptosis [3]. In modern medicine, the most common cancer therapy approaches include surgery, chemotherapy, and/or radiotherapy with a significant burden on the life quality of patients [4].

Breast cancer is characterized by the uncontrolled proliferation of epithelial cells in the breast tissue, with the potential to invade surrounding tissues and metastasize to distant organs with high prevalence worldwide [5]. Depending on the immunohistochemical expression of hormone receptors on the breast epithelial cell surface, breast cancer is classified into four groups: Estrogen Receptor (ER) or Progesterone Receptor (PR) positive (Luminal A and Luminal B), Human Epidermal Growth Factor Receptor 2 (HER2) positive (including ER and PR positivity) and Triple Negative Breast Cancer (TNBC) that is characterized by the absence of ER, PR and HER2 which turns those cells into highly pleomorphic and malignant cell types [6]. MDA-MB-231 breast cancer cell line is classified as TNBC, adherent, spindle-shaped cells with invasive properties with the absence of receptor expression at the cell surface which contributes to their aggressive nature and resistance to standard hormonal therapies [7, 8].

The resistance of breast cancer to different medical treatments has led researchers to develop targeted therapies [9]. The first targeted therapies were ER and PR inhibitors (tamoxifen) and anti-HER-2 therapies (Herceptin) targeting HER-2 positive molecular subtypes [10]. However, triple-negative requires a specific therapeutic approach [11]. Therefore, chemotherapeutic agents such as paclitaxel, doxorubicin, and cyclophosphamide are usually used as (neo) adjuvant systemic therapy for patients with TNBC, followed by radiotherapy [12]. However, their toxic effects on the normal, rapidly renewing cells (bone marrow cells), and their lack of specificity lead to many potentially fatal side effects, such as heart failure that has been linked to the use of these cytotoxic agents [13]. TNBC patients have a significantly lower five-year survival rate than hormone receptor-positive breast cancer patients, and there are few treatment options [14]. Due to this reason, in recent years, many scientists have been investigating bioactive compounds found in plants to develop novel therapeutics for cancer therapy, such as breast cancer [15].

Tecoma stans (L.) Juss. ex Kunth (Bignoniaceae) is an evergreen plant commonly known as yellow elder, campanilla, trumpet bush, and Esperanza [16]. Pharmacological studies have revealed that Tecoma stans possesses antioxidants, anti-tumor, antibacterial, and pain-relieving properties. These properties are attributed to identified and isolated compounds such as monoterpene alkaloids, phenolic acids, flavonoids, carotenoids, terpenoids, glycosides, phytosterols, volatile oils, and unsaturated fatty acids [17]. Pharmacological studies have highlighted the diverse bioactivities of Tecoma stans extracts such as a significant blood glucose-lowering effects in both in vitro and in vivo models, primarily attributed to bioactive compounds such as the alkaloid tecomine and the flavonoid chrysoeriol [18]. In addition to their antidiabetic potential, methanolic and ethanolic extracts of T. stans exhibit potent antioxidant activity, as evidenced by their DPPH and ABTS radical scavenging capacity, which is linked to their high phenolic and flavonoid content [19]. These antioxidant effects may contribute to the therapeutic profile of the plant in oxidative stress-related disorders. Furthermore, T. stans shows broad-spectrum antimicrobial activity, with extracts inhibiting pathogens such as Staphylococcus aureus, Escherichia coli, and Fusarium species, likely due to the presence of alkaloids, flavonoids, and terpenoids [20]. Anti-inflammatory activity has also been documented, particularly in chloroform root extracts that significantly reduced paw edema in rats by modulating inflammatory mediators [21]. Importantly, growing evidence supports the anticancer potential of T. stans extracts, which have exhibited cytotoxicity against breast (MDA-MB-231), prostate, and ovarian cancer cell lines. Compounds such as squalene and 2,3-dihydrofarnesol are believed to contribute to this effect through mechanisms that may involve apoptosis and cell cycle regulation [22]. For instance, methanolic leaf extract of Tecoma stans showed concentration-dependent cytotoxicity against weakly metastatic Luminal A type MCF-7 breast cancer and HeLa (cervical cancer) cells, with IC₅₀ values indicating moderate activity, possibly mediated through apoptosis induction and oxidative stress modulation [23]. Similarly, methanolic extracts of T. stans have shown notable cytotoxic effects against non-small cell lung cancer cell line A549, inhibiting viability. This strong inhibitory effect is probably due to induction of apoptotic pathway in the presence of T. stans phenolic bioactive molecules with known anticancer activity [24].

In this in vitro study, we aimed to investigate the phytochemical composition of Tecoma stans leaves and flowers (collected from Nicosia) methanolic extract (TSLME & TSFME) and their cytotoxic, anti-proliferative, anti-motile, and morphometric effects on highly metastatic triple negative breast cancer cell line, MDA-MB-231.

Methods

Plant collection and identification

Wild Tecoma stans samples were collected from Gönyeli 99,150 (Nicosia), Northern Cyprus in July 2021. Northern Cyprus does not require specific permission for collection of Tecoma stans from natural habitats. Plant samples were authenticated by Asst. Prof. Dr. Emmanuel Halilu; Chair of Pharmacognosy Department, Faculty of Pharmacy, Cyprus International University (CIU) as an Herbarium Specimen on 14/07/2021 with Voucher Number: CIU/ PHAR/ BIGN/ 001. In addition, one of the two specimens of the plant was deposited in the CIU Public Herbarium for future reference.

Preparation and Extraction

Fresh Tecoma stans leaves and flowers were washed with deionized water and then air-dried in the laboratory at room temperature. After grinding, weighing, and storing separately in sterilized containers, the leaves and flower powders were extracted with methanol (Merck, Germany) using the Soxhlet method, described by Thirumal et al. [18] with a few modifications. 30 g of leaf and flower powders were separately mixed with methanol (300 mL; 80% methanol: water) at 60 °C for 8 h and filtered through, Whatman filter paper N°1, concentrated via rotary evaporator (Rotary Evaporator, Heidolph, Germany) at 50 °C. Concentrated samples were weighed, labeled, and stored at 4 °C until further use. The percentage yield of extraction was calculated by the following formula:

$$\%Yield=\frac{WeightofDriedExtract}{WeightofDriedPlantSample}\times 100$$ 1

Gas Chromatography-Mass Spectrophotometry (GC–MS) Analysis

TSLME and TSFME were individually analyzed using GC–MS to study their bioactive constituents via GCMS-QP2010 Plus (Shimadzu, Japan) according to the method of Anburaj et al. [19] with slight modifications. The oven temperature was set from 40ºC (for 2 min) to 150ºC with a gradual increase of speed 8ºC/min, then to 250ºC at the same previous speed which ended with isothermal maintenance for 10 min at 300ºC. In addition, the gas chromatograph is interfaced with a mass spectrometer instrument with the following parameters: Teknokroma TRB-5MS column (diameter 0.25 µm, thickness 0.25 μm and length 30 m) which operates in electron impact mode at 70 eV with helium gas (99.999%) as carrier gas. The injection volume of 1 µL was used at an injector temperature of 270 ºC; an ion source temperature of 200 ºC with a mass spectrum between 35–500 amu and a constant helium flow rate of 1 mL /min. The total analysis time was 30 min/extract. Furthermore, compounds presented in the TSLME and TSFME were identified through the WILEY7.LIB MS software [25].

Fourier-transform infrared (FTIR) spectra of Tecoma stans leaf methanolic extract (TSLME) and flower methanolic extract (TSFME)

FTIR analysis was conducted to identify the functional groups present in the methanolic extracts of Tecoma stans leaves (TSLME) and flowers (TSFME), thereby providing insights into the classes of phytochemicals potentially responsible for their biological activities. The spectra were recorded in the range of 400 to 4500 cm⁻¹ using a Shimadzu FTIR Prestige-21 spectrophotometer [26].

Cell culture conditions of MDA-MB-231 breast cancer cell lines

Triple-negative highly metastatic human breast cancer cell line MDA-MB-231 was obtained from Imperial College London, UK (courtesy of Prof.Dr. Mustafa Djamgoz). Cells were grown in Dulbecco’s modified eagle medium (Gibco by Life Technologies™, USA), supplemented with 4 mmol/L L-glutamine (Gibco by Life Technologies™, USA), 5% fetal bovine serum (Gibco by Life Technologies™, USA) and penicillin/streptomycin (2%) (Gibco by Life Technologies™, USA) at 37 °C, 5% CO₂, and 95% humidity until cells reached 90–100% confluence [27].

Cytotoxicity study (Trypan Blue Exclusion Assay)

The trypan blue exclusion assay was performed to determine the cytotoxic effect of TSLME and TSFME on triple-negative MDA-MB-231 breast cancer cells. The cells were grown in 35 mm dishes (Thermo Scientific Nunc Cell Culture Dishes; Thermo Scientific; USA) (3 × 10⁴ cells/mL) and incubated for 24 h before being treated with different concentrations (800, 200, 50 µg/mL) of TSLME (A1, A2, A3), TSFME (B1, B2, B3). Negative control dishes received only cell culture media (DMEM control) and DMSO (D1: 0.053%, D2: 0.013%, D3: 0.003%; DMSO control: DMSO & cell culture media %) (Sigma Aldrich Inc., Germany) for 24, 48, and 72 h. After each treatment, the dishes were filled with trypan blue dye (4%) (Gibco by Life Technologies™, USA) and incubated for 15 min before observing and counting the number of viable cells in each dish from 30 randomly selected areas under an inverted microscope (Leica, Germany) at X20 magnification [28].

Cell proliferation study (MTT Assay)

Antiproliferative effects of TSLME and TSFME on MDA-MB-231 triple-negative breast cancer cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as previously described by Fraser et al. [29]. The cells were plated and incubated in 96-well plates (1 × 10⁴ cells/ well) after being treated with various concentrations (200, 100, 50 µg/mL) of TSLME (A2, A4, A3), TSFME (B2, B4, B3) for 24, 48, and 72 h. Negative control wells received only cell culture media (DMEM control) and DMSO (D2: 0.013%, D4: 0.0065%, D3: 0.003%; DMSO control: DMSO & cell culture media %). After each treatment, cells were incubated with MTT solution which was subsequently removed. 200 µL of DMSO (100%) was added and the absorbance was measured at a wavelength of 490 nm using the Absorbance Microplate Reader (ELx800, Biotek Instruments, Labx; Canada). Treated MDA-MB-231 breast cancer cell proliferation was calculated by normalizing to the 0 h plates and the corresponding DMSO control group.

Wound healing study assay (Lateral Motility Assay)

Wound Healing Assay was assessed to evaluate the inhibitory effects of different concentrations (50, 25, 15 µg/mL) of TSLME (A3, A5, A6), TSFME (B3, B5, B6) on breast cancer cell motility [21]. Negative control plates received only cell culture media (DMEM control) and DMSO (D3: 0.003%, D5: 0.0015%, D6: 0.001%; DMSO control: DMSO & cell culture media %). The cells were plated in 35 mm dishes (1 × 10⁶ cells/mL) which fifteen vertically parallel lines were drawn using a fine (blue) marker and incubated for 24 h at 37 °C and 5% CO₂. After incubation, three wounds were created (three horizontal parallel lines) using 200 µL Gilson pipette tips, and the contents were rinsed three times with 1000 µL cell culture media (DMEM) to remove floating cells before being treated for 24 and 48 h. Furthermore, of the 45 points of intersection between the horizontal lines and vertical wounds that were created/dish, 25 points were photographed (scale:200 µm) per dish after 0, 24, and 48 h at X20 magnification by using a digital camera connected to the inverted microscope (Leica, Germany). The distance of the wounds was measured and analyzed with ImageJ software, and the lateral motility was calculated using the motility index (MoI) following the formula:

$$MoI=1-\left(\frac{\mathit{Wt}}{\mathit{Wo}}\right)$$ 2

Wt represents the wound's width at a specific time (24 or 48 h) and Wo represents the initial width at zero hour (0 h) [29].

Morphological study (Morphometric Analysis)

Morphometric analysis of MDA-MB-231 cells upon TSLME and TSFME treatment was performed according to Fraser and coworkers [30]. The cells were plated 35 mm sterile culture dish (3 × 10⁴ cells/mL) and incubated for 24 h. After treatment with different concentrations (800, 200, 50 µg/mL) of TSLME & TSFME, for 24, 48, and 72 h, the cells were visualized at X20 magnification under an inverted microscope (Leica, Germany) and analyzed using Image J software. During this visualization, present Bipolar cells were randomly selected, and photographed (scale: 100 µm). Morphometric parameters such as field diameter (FD), cell body diameter (CBD), and process thickness (PT) (Fig. 1) were used [30] in order to assess the morphometric effects of TSLME and TSFME.

Fig. 1.

Schematic illustration of a bipolar cell with morphological indices showing the measured parameters. Field diameter (FD), cell body diameter (CBD), process thickness (PT), and process length (PL). (modified from Fraser et al. [30])

Statistical analysis

All experiments were carried out with a minimum of three times in triplicates and data are represented as means ± standard errors of the mean (SEM) (n ≥ 3). Graphical representations and statistical analysis were performed. The paired or unpaired student’s t-test, as appropriate, was used to establish pairwise statistical significance. (X) p > 0.05 was considered non-significant; (*) p < 0.05 was considered significant; (**) p < 0.01 was considered slightly significant and (***) p < 0.0001 highly significant.

Results

Extraction Yield of Tecoma stans methanolic extracts

The percentage yield of methanolic extract of Tecoma stans leaves and flowers were calculated after concentration with rotary evaporator where yield for TSLME and TSFME was 41.8% and 41%, respectively (Table 1).

Table 1.

Percentage yield of Tecoma stans methanolic leaf (TSLME) and flower (TSFME) extracts

Sample (30 g)	Plant material (gr)	% extract yield
TSLME	12.54	41.8%
TSFME	12.3	41%

Thus, the results obtained are in line with research conducted by Chatha et al. [31] who demonstrated that the maximum yield of rice bran extract was obtained with 80% methanol (aqueous methanol) as the solvent used. Furthermore, the yield of TSLME is slightly higher (0.8% of difference) than TSFME. The differences in yields between all of them might be attributed to the presence of several extractable compounds in different parts of the plant resulting from the phytochemical compositions that vary according to the plant or between different parts of the plant [32].

Qualitative analysis of Tecoma stans extracts

The results from GC–MS analysis indicated that TSLME (Fig. 2A) and TSFME (Fig. 2B) showed the presence of 40 and 39 main peaks, respectively. Both TSLME and TSFME extracts have 15 peaks corresponding to same bioactive molecules with antimicrobial (AM), antibacterial (AB), antitumoral (AT), hepatoprotective (HP), antioxidant (AO), chemo-preventive (CP), antiproliferative (AP), antifungal (AFG), anti-inflammatory (AIF), Immunomodulatory (IMD) effects (Tables 2 and 3).

Fig. 2.

GC–MS chromatogram of methanolic extracts of leaves (A) and flowers (B) of Tecoma stans. 40 (TSLME) and 39 (TSFME) peaks were mainly selected from the chromatogram of each extract to be identified and listed respectively in Tables 2 and 3 the bioactive compounds/peak present in each extract

Table 2.

Pharmacological Activities of bioactive compounds (main peaks selected) present in TSLME

Peak	Compound Name	Retention Time (Min)	Area (%)	Pharmacological Activities (Reference)
1	Cyclopentadiene, 1,2,3,4,5-pentamethyl-	6.107	0.21	NAR
2	Decane	7.641	0.54	AM [33]
3	Vanillin	8.639	0.34	AO, AIF, NRP, AC [34–36]
4	Phenethylamine, 3,4,5-trimethoxy- α-methyl-	9.012	0.17	NAR
5	Cyclopentasiloxane, decamethyl	10.241	0.20	AM [37]
6	3,4-Dihydroxyphenol ethanol ( 3-hydroxytyrosol)	11.683	0.18	AO, NRP [38, 39]; AT, AP [40–42]; AM [43]
7	Cyclohexasiloxane, dodecamethyl	12.897	0.24	AM [44]; AFG [45]; AB, AFL, IMD and AT [46]
8	Mandelic acid, 3,4-dihydroxy, (4TMS)	14.031	0.42	AM,AO [47, 48]
9	Cholan-12-one, 3-(acetyloxy)-, (3α. 5β.)-	14.958	0.30	NAR
10	Cycloheptasiloxane, tetradecamethyl	15.301	0.59	AM [44]; AFG [45]; AB, AFL, IMD and AT [46]
11	1-Bromotetradecane ( Myristyl bromide)	15.561	0.44	NAR
12	Cyclododecasiloxane, tetracosamethyl	16.276	6.27	AFG [45];HP, ASP, ARH [49]
13	1,2-Benzenedicarboxylic acid, diethyl ester	17.256	6.85	AM, AFG, AMR [50]; AC, CT [51]
14	2-Cyclohepten-1-one ( Tropilene)	17.832	1.39	NAR
15	1,1,3,3,5,5,7,7,9,9,11,11Dodecamethylhexasiloxane	18.279	2.22	AM, AST [53]
16	Cyclooctasiloxane, hexadecamethyl	18.646	0.75	AM [44]
17	D-Xylitol	18.850	1.06	NAR
18	9-Octadecenoic acid, 2-[(trimethylsilyl)oxy]−1-[[(trimethylsilyl)oxy]methyl]ethyl ester	19.189	0.83	AC, HP [54]
19	1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-Hexadecamethyl-Octasiloxane	20.258	1.92	AM [55]
20	Cyclodecasiloxane, eicosamethyl-	20.845	3.30	AM, AHT, AO [55]
21	Iron, monocarbonyl-(1,3-butadiene-1,4-dicarbonic acid, diethyl ester), a, a’-dipyridyl	21.244	1.01	NAR
22	6,10-Dodecadien-3-ol, 3,7,11-trimethyl- ( 2,3-Dihydrofarnesol)	21.576	3.34	NAR
23	2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl- ( Squalene)	21.969	1.33	AB, AO, AC, CP [56–59]
24	1H-Purin-6-amine, [(2-fluorophenyl)methyl]-	22.381	4.77	AO [60]
25	5-Pregnene-3β,20β,21-triol TMS	22.815	1.01	NAR
26	Imidazo[4,5,1-jk][1, 4]benzodiazepin-7(4H)-one, 2-[4-[(dimethylamino)methyl]phenyl]−5,6-dihydro-	23.575	1.15	NAR
27	Ethyl 1-hexyl-4-hydroxy-2(1H)-oxo-3 quinolinecarboxylate	23.927	0.62	NAR
28	3-Chlorodecanoic acid, chloromethyl ester	24.207	0.33	NAR
29	Purine, 6-(3-methyl-2-butenyl)amino, TMS	24.920	0.32	AP [61]
30	Cyclononasiloxane, octadecamethyl	25.843	4.38	AFG [62]; AO [63, 64]

AM Antimicrobial, AB Antibacterial, AT Antitumor, HP Hepatoprotective, AO Antioxidant, AC Anticancer, NAR No Activity Reported, CP Chemopreventive, AP Antiproliferative, AFG Antifungal, AFL Antifouling, AIF Anti-inflammatory, IM )Immunomodulatory, ASP Antispasmodic, ART Antirheumatic, AMR Antimalarial, CT Cytotoxicity, AHT Anthelmintic, NRP Neuroprotective, AST Antiseptic

Table 3.

Pharmacological Activities of bioactive compounds (main peaks selected) present in TSFME

Peak	Compound Name of TSFME GC–MS	R.Time (Min)	Area (%)	Pharmacological Activities (Reference)
1	Cyclopentadiene, 1,2,3,4,5-pentamethyl-	6.079	0.21	NAR
2	Decane	7.633	1.07	AM [33]
3	Vanillin	8.644	0.55	AO, AIF, NRP, AC [34–36]
4	Benzeneacetic acid, 3-methoxy-α,4-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester	9.022	0.28	AC, AFG, AB [65, 66]
5	Cyclopentasiloxane, decamethyl	10.246	0.29	AM [37]
6	1,3,5,7,9-Pentamethyl Cyclopentasiloxane	11.685	0.33	NAR
7	Cyclohexasiloxane, dodecamethyl	12.897	0.47	AM [44]; AFG [45];AB, AFL, IMD and AT [47]
8	2H-1,4-Benzodiazepin-2-one, 7-chloro-5-(2-fluorophenyl)−1,3-dihydro-3-hydroxy-	14.074	0.36	NAR
9	Cycloheptasiloxane, tetradecamethyl	15.304	0.56	AM [44]; AFG [45];AB, AFL, IMD and AT [46]
10	Pentasiloxane, 1,1,3,3,5,5,7,7,9,9-decamethyl-	16.207	2.16	NAR
11	1,2-Benzenedicarboxylic acid, diethyl ester	17.228	12.50	AM, AFG, AMR [50]; AC, CT [51, 52]
12	3,3-Dimethyl-2-phenyl-2-(1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl) azirane	17.750	2.90	NAR
13	n-Octacosane	18.001	0.92	AT [67], AO, WHP [68]
14	1,1,3,3,5,5,7,7,9,9,11,11Dodecamethylhexasiloxane	18.278	3.15	AM, AST [53]
15	2,6-Dihydroxybenzoic acid, 3TMS derivative	18.640	1.04	AM, AO, AC, CP [69]
16	3,3-Dimethyl-2-butanone	18.843	0.99	NAR
17	Ethyl 1-hexyl-4-hydroxy-2(1H)-oxo-3 quinolinecarboxylate	19.149	1.29	NAR
18	Acetophenone, 4'-methoxy-3'-methyl-	19.364	0.90	NAR
19	Glycine, N-[[(4-methoxyphenyl)methoxy]carbonyl]-N-methyl-	19.591	1.48	NAR
20	Cyclononasiloxane, octadecamethyl	19.876	1.49	AFG [62]; AO [63, 64]
21	3,3-Dimethyl-2-butanone	20.259	1.11	NAR
22	2-(4,5-Dihydro-3-methyl-5-oxo-1-phenyl-4-pyrazolyl)−5-nitrobenzoic acid	20.511	0.83	AT [63]
23	Cyclododecasiloxane, tetracosamethyl	21.039	2.79	AFG [45];HP, ASP, ARH [49]
24	Norepinephrine, (R)-, 4TMS derivative	21.878	0.80	NAR
25	Cyclodecasiloxane, eicosamethyl-	22.437	0.92	AM, AHT, AO [49]
26	9,12-Octadecadienoic acid (Z,Z)-, 2,3-bis[(trimethylsilyl)oxy]propyl ester	22.978	0.42	AF, AB, AT, AIF, HP [70–72]
27	1H-Purin-6-amine, [(2-fluorophenyl)methyl]-	23.359	0.94	AO [60]
28	5-Pregnene-3β,20β,21-triol TMS	25.189	0.43	NAR
29	1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-Hexadecamethyl-Octasiloxane	25.463	1.37	AM [55]

AM Antimicrobial, AB Antibacterial, AT Antitumor, HP Hepatoprotective, AO Antioxidant, AC Anticancer, NAR No Activity Reported, CP Chemopreventive, AP Antiproliferative, AFG Antifungal, AFL Antifouling, AIF Anti-inflammatory, IMD Immunomodulatory, ASP Antispasmodic, ART Antirheumatic, AMR Antimalarial, CT Cytotoxicity, AHT Anthelmintic, NRP Neuroprotective, AST Antiseptic

Bioactive molecules presented with higher intensities in TSLME compared to TSFME were listed such as Cyclododecasiloxane, tetracosamethyl (TSLME: 6.27% > TSFME: 2.79%); 1H-Purin-6-amine, [(2-fluorophenyl)methyl]- (TSLME: 4.77% > TSFME: 0. 94%); Cyclononasiloxane, octadecamethyl (TSLME: 4.38% > TSFME: 1.49%); Cyclodecasiloxane, eicosamethyl- (TSLME: 3.30% > TSFME: 0. 92%); 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-Hexadecamethyl-octasiloxane (TSLME:1.92% > TSFME: 1.37%); 5 Pregnene-3β,20β,21-triol TMS (TSLME: 1.01% > TSFME: 0.43%). Other detected compounds showed lower peak intensities in TSLME compared to TSFME, including: 1,2 Benzenedicarboxylic acid, diethyl ester (TSFME:12.50% > TSLME:6.85%); 1,1,3,3,5,5,7,7,9,9,11,11Dodecamethylhexasiloxane (TSFME: 3.15% > TSLME: 2. 22%); Ethyl 1-hexyl-4-hydroxy-2(1H)-oxo-3 quinolinecarboxylate (TSFME: 1.29% > TSLME: 0.62%); Decane (TSFME: 1.07% > TSLME: 0.54%).

However, bioactive compounds specific to each extract were identified with significant peak intensities including for TSLME: 6,10-Dodecadien-3-ol, 3,7,11-trimethyl- (3.34%); 2-Cyclohepten-1-one (Tropilene) (1.39%); 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl- (Squalene) (1. 33%); Imidazo[4,5,1-jk] [1, 4]benzodiazepin-7(4H)-one, 2-[4 [(dimethylamino) methyl]phenyl]−5,6-dihydro- (1.15%); D-xylitol (1.06%); Iron, monocarbonyl-(1,3-butadiene-1,4-dicarbonic acid, diethyl ester), a, a'-dipyridyl (1.01%) and for TSFME: 3,3-Dimethyl-2-phenyl-2-(1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl) azirane (2.90%); Pentasiloxane, 1,1,3,3,5,5,7,7,9,9-desmethyl- (2. 16%); Glycine, N-[[(4 methoxyphenyl)methoxy]carbonyl]-N-methyl- (1.48%); 3,3-Dimethyl-2-butanone (1.11%); 2,6-Dihydroxybenzoic acid, 3TMS derivative (1.04%) (Tables 2 and 3).

Fourier-transform infrared (FTIR) spectra of Tecoma stans leaf methanolic extract (TSLME) and flower methanolic extract (TSFME)

Based on the peak values ​​in the infrared radiation region, the functional groups of the active compounds in the TSLME and TSFME were determined using the FTIR spectrum. Figure 3 and Table 4 present these analyses. Nine peaks with the following wavenumbers were present in the FTIR spectra and were shared by both extracts (TSLME and TSFME): 2947.23 cm⁻¹, 2831.50 cm⁻¹, 1651.06 cm⁻¹, 1450.46 cm⁻¹, 1110.99 cm⁻¹, 1018.41 cm⁻¹, 686.65 cm⁻¹, 663.51 cm⁻¹, 1201.79 cm⁻¹. Four distinct peaks were visible in the TSLME FTIR spectra at the wavenumbers of 3356.13 cm⁻¹, 1404.17 cm⁻¹, 486.06 cm⁻¹, and 470.63 cm⁻¹. Last but not least, the TSFME FTIR spectra revealed three distinct peaks at wave numbers 3348.42 cm⁻¹, 1411.89 cm⁻¹, and 493.77 cm⁻¹.

Fig. 3.

FTIR spectrum study of methanolic crude extracts of leaves (A) and flowers (B) of Tecoma stans. The functional groups identified in the 13 (TSLME) and 12 (TSFME) peaks, primarily selected from spectroscopy, are listed in Table 4, respectively

Table 4.

Identification of functional groups present in TSLME and TSFME by FTIR spectrum

Wavenumbers (cm − 1)	TSLME [ Y (%T)]	TSFME [ Y (%T)]	Bond	Functional groups
470.63	95.35	-	C-I stretch	Alkyl halides
486.06	94.67	-	C-I stretch	Alkyl halides
493.77	-	93.84	C-I stretch	Alkyl halides
601.79	82.07	81.88	C–Br stretch	Alkyl halides
663.51	79.09	79.40	C–Br stretch	Alkyl halides
686.65	79.46	79.77	= C-H bend	Alkenes
1018.41	61.19	60.46	C-O stretch	Alcohols, carboxylic acids, esters, ethers
1110.99	93.40	93.34	C-O stretch	Alcohols, carboxylic acids, esters, ethers
1404.17	92.95	-	C–C stretch (in-ring)	Aromatic
1411.89	-	92.84	C–C stretch (in-ring)	Aromatic
1450.46	93.39	93.36	S = O stretch, C-H bend	Sulfate, Alkanes
1651.06	94.35	94.70	-C = C- stretch	Alkenes
2831.50	92.98	92.78	C-H stretch, = C-H Stretch, C = 0 stretch, N–H stretch	Alkanes, Aldehydes, ketones, Amines
2947.23	91.38	91.25	O–H stretch, C-H stretch, N–H stretch	Carboxylic acids, Alkanes, Amines
3348.42	-	82.83	O–H stretch, H-bonded	Alcohols, phenols
3356.13	82.77	-	O–H stretch, H-bonded	Alcohols, phenols

The bands at wavenumbers 3348.42 cm⁻¹ and 3356.13 cm⁻¹ correspond to the O–H stretch and H-bonded O–H, which are characteristic of the presence of alcohols and phenols. The O–H, C-H, and N–H stretches, which stand for carboxylic acids, alkanes, and amines, respectively, are peculiar to the band at 2947.23 cm⁻¹. C-H, = C-H, C = 0, and N–H stretches that correspond to alkanes, aldehydes, ketones, and amines are represented by the subsequent peak at 2831.50 cm⁻¹. Alkenes are identified by the presence of the -C = C- stretch, as indicated by the band at 1651.06 cm⁻¹. The band represents the sulfate and alkane group at 1450.46 cm⁻¹, which is unique to the S = O stretch and C-H bend. Specific to the C–C stretch (in-ring), the bands at 1404.17 cm⁻¹ and 1411.89 cm⁻¹ indicate an aromatic compound. Alcohols, carboxylic acids, esters, and ethers are represented by the bands at 1018.41 cm⁻¹ and 1110.99 cm⁻¹ wavenumbers, which correspond to the C-O stretch. The proposed alkenes have a = C-H bend, represented by the peak at 686.65 cm⁻¹. C–Br stretch is seen in the peaks at 601.79 cm⁻¹ and 663.51 cm⁻¹, indicating alkyl halides. Furthermore, alkyl halides are represented by the C-I stretch, which is indicated by the bands at 486.06 cm⁻¹, 470.63 cm⁻¹, and 493.77 cm⁻¹ wavenumbers.

Cytotoxic effects of Tecoma stans flower and leaf extracts (TSLME & TSFME) on cell viability

Trypan Blue assay was conducted in order to assess cytotoxic effects of Tecoma stans flower and leaf methanolic extracts on MDA-MB-231 breast cancer cells. The higher concentrations (A1/B1: 800 µg/mL) demonstrated significant cytotoxic effect compared to the control experiment (D1: 0.053%) (p < 0.0001; Fig. 4A and 4B) on MDA-MB-231 cells with a more robust cell death (%) in TSLME (24 h: 22.54%; 48 h: 62.95%; 72 h: 98.46%) compared to TSFME (24 h: 0.63%; 48 h: 0.93%; 72 h: 1. 25%). TSLME has a significant cytotoxic effect compared to the TSFME at 800 µg/mL at all tested time points. However, at 50 µg/mL (A3/B3) and 200 µg/mL (A2/B2) concentrations, no cytotoxic effect was observed (p > 0.05; Fig. 3A and 3B) in both extracts compared to the DMSO controls (D3: 0.003%, D2: 0.013%; no cytotoxic effect was observed compared to DMEM control) for all tested timeline (Fig. 4A and 4B) [73, 74].

Fig. 4.

TSLME (A) and TSFME (B) exerted a significant cytotoxic effect on highly metastatic breast cancer cell line MDA-MB-231 upon 24, 48, and 72 h of incubation at 800 µg/mL (TSLME/TSFME) concentration. 0.053% (D1), 0.013% (D2), and 0.003% (D3) of DMSO controls were used against each plant extract concentration, respectively (800 µg/mL, 200 µg/mL, and 50 µg/mL). Data are represented as means ± SEM (n ≥ 3). Significance: (X) p > 0.05; (***) p < 0.0001

Antiproliferative effects of TSLME and TSFME

Effects of TSLME and TSFME on MDA-MB-231 cell proliferation were assessed via MTT assay in concentration-dependent manner [50 (A3, B3, D3), 100 (A4, B4, D4) and 200 µg/mL (A2, B2, D2)]. Those concentrations were chosen where there was no cytotoxic effect on MDA-MB-231 cells. Upon 24 h incubations, no inhibition of MDA-MB-231 cell proliferation was recorded in all tested concentrations of TSLME and TSFME (p > 0.05; Fig. 5A and B)0.48 and 72 h’ incubations of MDA-MB-231 cells with TSLME and TSFME significantly inhibited breast cancer cell proliferation, particularly at 100 µg/mL (A4/B4) and 200 µg/mL (A2/B2) compared to negative control experiments (D4: 0.0065%, D2: 0.013%) (p < 0. 01; Fig. 4A and B). On the other hand, the number of normalized viable cells values at 72 h obtained from MTT assay results suggests that the antiproliferative effect of Tecoma stans on MDA-MB-231 cells was more robust at 100 and 200 μg/mL upon incubations with TSFME compared to TSLME [(TSLME A4: 1.38 ± 0.06 & TSFME B4: 1.28 ± 0.05), (TSLME A2: 1.37 ± 0.09 & TSFME B2: 1.07 ± 0.32)] at 72 h. However, at 50 µg/mL (A3/B3) both TSFME and TSLME showed no inhibition (p > 0.05; Fig. 5A and B) on breast cancer cell proliferation due to very low concentrations of bioactive compounds (singly and/or synergistically) against metastatic cells proliferation.

Fig. 5.

TSLME (A) and TSFME (B) extracts demonstrated a significant antiproliferative effect against MDA-MB-231 cell line upon incubations for 48 and 72 h. 0.013% (D2), 0.006% (D4), and 0.003% (D3) of DMSO controls were used against each plant extract concentration, respectively (200 µg/mL, 100 µg/mL, and 50 µg/mL). Data are represented as means ± SEM (n ≥ 3). Significance: (X) p > 0.05; (*) p < 0.05; (**) p < 0.01

Effects of TSLME and TSFME on Lateral Motility of Metastatic Cells

The lateral motility assay (the wound healing assay) was performed to evaluate the inhibitory effect of TSLME and TSFME on the motility (migration) of highly metastatic MDA-MB-231 breast cancer cell lines. 15 (A6, B6, D6), 25 (A5, B5, D5), and 50 μg/mL (A3, B3, D3) were chosen where there was no significant antiproliferative and cytotoxic effect on MDA-MB-231 breast cancer cells. Motility assay results revealed that [Fig. 6D (TSLME) and 6E (TSFME)] upon 24 h incubations, TSLME and TSFME have a significant anti-motile effect (p < 0.05; Fig. 6D and E) on MDA-MB-231 cells compared to their negative control experiments (25 μg/mL MoI: TSLME A5: 0.46 ± 0.04 & TSFME B5: 0.45 ± 0.04; 50 μg/mL MoI: TSLME A3: 0.38 ± 0.04 & TSFME B3: 0.39 ± 0.06). On the other hand, despite the increase of MDA-MB231 cells' motility index upon 48 h of incubations, only the 50 μg/mL concentration (50 μg/mL MoI: TSLME A3: 0.65 ± 0.03 & TSFME B3: 0.70 ± 0.01) in both extracts showed significant anti-motile effect (p < 0.01; Fig. 6D and E) compared to the negative control experiments. However, no motility was observed at 50 μg/mL concentration (A3/B3) upon 72 h (p < 0.01; Fig. 6D and E) incubations compared to the control experiments (motility index of TSLME/TSFME <  < D3: 0.003%) which might be the evidence of chemoresistance of MDA-MB-231 cells towards both extracts. Similarly, at 15 µg/mL (A6/B6) concentration, both TSLME and TSFME had no inhibitory effect on MDA-MB-231 cell motility compared to control experiments for all tested timelines (p > 0.05; Fig. 6D and E).

Fig. 6.

Effects of TSLME and TSFME on the lateral motility of MDA-MB-231 cells. MDA-MB-231 cell lines motility (A, B, C) was inhibited by TSLME (D) and TSFME (E) at 24 h and 48 h’ incubations. (Scale bar:200 µm; applicable to all panels). 0.003% (D3), 0.0015% (D5), and 0.0009% (D6) of DMSO controls were used against each plant extract concentration, respectively (50 µg/mL, 25 µg/mL, and 15 µg/mL). Data was presented as means ± SEM (n ≥ 25). Significance: (X) p > 0.05; (*) p < 0.05; (**) p < 0.01

Effects of TSLME and TSFME on bipolar cell morphological development

Effects of TSLME and TSFME on MDA-MB-231 cell morphology were evaluated by measuring parameters such as cell body diameter (CBD), field diameter (FD), and the process thickness (PT). Three concentrations of TSLME and TSFME were used [50 (A3, B3), 200 (A2, B2), and 800 μg/mL (A1, B1)] to assess the effects of extracts on bipolar breast cancer cell morphology [Fig. 7A (TSLME) and Fig. 8A (TSFME)] upon 24, 48 and 72-h incubations.

Fig. 7.

The morphological changes of the MDA-MB-231 cells (A) upon treatment with TSLME (50, 200 and 800 µg/mL) were observed using an inverted microscope (X 20 magnification) at 24, 48 and 72 h incubations. TSLME significantly reduced MDA-MB-231 breast cancer Body Diameter (B), Field Diameter (C), and the Thickness Process (D) upon 24, 48, and 72 h of incubation. Data are presented as means ± SEM (n ≥ 3). Significance: (X) p > 0.05; (*) p < 0.05; (**) p < 0.01; (***) p < 0.0001

Fig. 8.

The morphological changes of the MDA-MB-231 cells (A) upon treatment with TSFME (50, 200 and 800 µg/mL) were observed using an inverted microscope (X 20 magnification) at 24, 48 and 72 h incubations. TSFME significantly reduced MDA-MB-231 breast cancer Body Diameter (B), Field Diameter (C), and the Thickness Process (D) upon 24, 48, and 72 h of incubation. Data are presented as means ± SEM (n ≥ 3). Significance: (X) p > 0.05; (*) p < 0.05; (**) p < 0.01; (***) p < 0.0001

Cell body diameter of MDA-MB-231 cells (Figs. 7B and 8B) was significantly reduced upon incubations with TSLME and TSFME at 200 and 800 μg/mL concentrations in both concentration and time-dependent manner. TSLME treated MDA-MB-231 breast cancer cell body diameters were measured as follows: A1-800 μg/mL: 48.97% ± 1.94 (p < 0.0001) for 24 h; A2-200 μg/mL:76.51% ± 5.5 (p < 0.01), A1-800 μg/mL:39.27% ± 2.46 (p < 0.0001) for 48 h and A3-50 μg/mL:77.16% ± 8.8 (p < 0.05), A2-200 μg/mL:56.38% ± 1.12 (p < 0.0001), A1-800 μg/mL:0% (p < 0.0001) for 72 h incubation. On the other hand, TSFME treated MDA-MB-231 breast cancer cell body diameters were measured as follows: B2-200 μg/mL:83.49% ± 5.67 (p < 0.05), B1-800 μg/mL:55.76% ± 3.34 (p < 0.0001) for 24 h; B2-200 μg/mL:67.50% ± 4.67 (p < 0.01), B1-800 μg/mL:49.38% ± 3.38 (p < 0.0001) for 48 h and B3-50 μg/mL:79.98% ± 6.58 (p < 0.05), B2-200 μg/mL:42.33% ± 4.13 (p < 0.0001), B1-800 μg/mL:31.78% ± 3.01 (p < 0.0001) for 72 h incubation. These results collectively indicate that TSLME-treated cells at 800 μg/mL had a more robust effect on the overall reduction of the cell body diameter than the TSFME-treated cells for all tested time points.

TSLME treated MDA-MB-231 breast cancer cell field diameters were measured as follows: A1-800 μg/mL: 60.64% ± 1.46 (p < 0.0001) for 24 h; A2-200 μg/mL:68.35% ± 2.53 (p < 0.01), A1-800 μg/mL:41.56% ± 3.5 (p < 0.0001) for 48 h and A3-50 μg/mL:71.91% ± 7.21 (p < 0.01), A2-200 μg/mL:61.30% ± 12.8 (p < 0.05), A1-800 μg/mL:0% (p < 0.0001) for 72 h incubation. On the other hand, TSFME treated MDA-MB-231 breast cancer cell field diameters were measured as follows: B1-800 μg/mL:74.72% ± 6.12 (p < 0.01) for 24 h; B3-50 μg/mL:53.98% ± 13.45 (p < 0.05), B2-200 μg/mL:68.0% ± 5.74 (p < 0.01), B1-800 μg/mL:52.52% ± 11.44 (p < 0.01) for 48 h and B3-50 μg/mL:54.20% ± 9.74 (p < 0.001), B2-200 μg/mL:49.72% ± 5.37 (p < 0.01), B1-800 μg/mL:38.36% ± 4.89 (p < 0.01) for 72 h incubation. The field diameter (FD) of MDA-MB-231 cells (Figs. 7C and 8C) was reduced highly significantly by two extracts at 800 μg/mL, especially after incubations at 72 h of treatment [ TSLME (p < 0.0001) > TSFME (p < 0.01)].

TSLME treated MDA-MB-231 breast cancer cell process thickness were measured as follows: A2-200 μg/mL:55.66% ± 17.73 (p < 0.05), A1-800 μg/mL:47.91% ± 1.12 (p < 0.0001) for 24 h; A2-200 μg/mL:50.62% ± 6.47 (p < 0.0001), A1-800 μg/mL:28.53% ± 3.47 (p < 0.0001) for 48 h and A3-50 μg/mL:70.23 ± 2.55 (p < 0.01), A2-200 μg/mL:33.08% ± 9.55 (p < 0.05), A1-800 μg/mL:0%(p < 0.0001) for 72 h incubation. On the other hand, TSFME treated MDA-MB-231 breast cancer cell process thickness were measured as follows: B1-800 μg/mL:67.69% ± 3.56 (p < 0.01) for 24 h; B2-200 μg/mL:51.69% ± 5.16 (p < 0.01), B1-800 μg/mL:39.79% ± 4.5 (p < 0.0001) for 48 h and B3-50 μg/mL:56.12% ± 10.43 (p < 0.01), B2-200 μg/mL:43.72% ± 4.67 (p < 0.01), B1-800 μg/mL:28.93% ± 2.71 (p < 0.0001) for 72 h incubation. The size and overall shape of the process thickness (PT) of MDA-MB-231 cells upon TSLME and TSFME incubations were significantly reduced in both concentration and time-dependent manner.

However, the cell surface area of MDA-MB-231 cells that have been decreased after incubation at high (800 µg/mL) and low (50 µg/mL) concentrations for 48 h (Fig. 9A) compared with the DMSO control, were calculated based on measurements obtained from bipolar cells randomly selected in triplicate. So, it was demonstrated that cell surface area decrease was significantly higher on TSLME (p < 0.01; Fig. 9B) treated cells compared to the TSFME (Fig. 8C) treated ones (TSLME A1: 800 µg/mL; 60.27% ± 1.16 and TSLME A3: 50 µg/mL; 43.38% ± 2.28; TSFME B1: 800 µg/mL; 61.19% ± 2.99 and TSFME B3: 50 µg/mL; 35.16% ± 6.63).

Fig. 9.

Cells treated with TSLME and TSFME for 48 h (A) demonstrated morphological changes such as membrane blebbing (black arrows), nuclear condensation (yellow arrows), and the formation of apoptotic bodies (red arrows) and significant cell surface area decrease upon incubations by TSLME (D) and TSFME (E) in a concentration-dependent manner. Data are represented as means ± SEM (n ≥ 3). Significance: (*) p < 0.05; (**) p < 0.01

Discussion

The present study investigated the cytotoxic potential of Tecoma stans leaf and flower methanolic extracts (TSLME and TSFME, respectively) on MDA-MB-231 breast cancer cells. The results demonstrated that TSLME exhibited significantly higher cytotoxic activity compared to TSFME. This difference can be explained by the unique phytochemical profiles of each extract, which likely contribute to their distinct biological effects. In this section, we discuss the potential mechanisms behind the observed cytotoxicity, supported by previously reported bioactive compounds and recent literature.

Higher cytotoxic activity exhibited by TSLME compared to TSFME might be explained by presence of 2-Cyclohepten-1-one (Tropilene); 6,10-Dodecadien-3-ol, 3,7,11-trimethyl- (2,3-Dihydrofarnesol); 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl- (Squalene) (Table 2) where they singularly or synergistically exert higher cytotoxicity on MDA-MB-231 cells [75, 76]. Moreover, higher cytotoxicity exerted by TSLME compared to TSFME may also be due to the presence of the high intensity of some antioxidant compounds including Cyclononasiloxane octadecamethyl [63, 64], Cyclodecasiloxane eicosamethyl which was previously shown exhibit cytotoxic activity, particularly against cancer cell lines like MCF7, A2780, and HT29 [49] and 1H-purin-6-amine, [(2-fluorophenyl)methyl] (Table 2) [60] which may contribute breast cancer cell cytotoxicity along with the other bioactive compounds [77]. On the other hand; TSFME cytotoxicity can be attributed to the presence of the phenolic acid: 2,6-Dihydroxybenzoic acid, 3TMS derivative (Table 3). This compound was previously shown to exert a cytotoxic effect on MDA-MB-231 cells (IC₅₀: 3.97 ± 2.54) through the induction of apoptosis [69].

This antiproliferative activity is probably due to the presence of 1,2-Benzenedicarboxylic acid, diethyl in both TSLME and TSFME. Several in vitro studies previously demonstrated that 1,2-Benzenedicarboxylic acid, diethyl ester has an inhibitory effect on the proliferation of both MCF-7 and MDA-MB-231 cells [78, 79]. The inhibition of MDA-MB-231 cell proliferation by TSLME might also be explained by a synergistic effect between two of these specific compounds: 3,4-Dihydroxyphenol ethanol (3-hydroxytyrosol) and 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl- (Squalene) (Table 2). It was previously shown that Squalene combined with hydroxytyrosol have antitumoral activity on MDA-MB-231 cells which demonstrated that they inhibited the proliferation of MDA-MB-231 cells at 100 µM by promoting apoptosis and DNA damage [58].

Motility assay results demonstrated inhibitory effects of both tested extracts (TSLME and TSFME) on triple-negative breast cancer cell motility (Fig. 6D and 6E) at 25 and 50 μg/mL concentrations upon 24 h incubation. This phenomenon can be explained by the presence of the same compound such as vanillin in both extracts which was previously shown to demonstrate an anti-metastatic effect by inhibiting the invasion and migration of breast cancer cells through the deactivation of the enzymatic activity of matrix metalloproteinase-9 (MMP-9) in vivo and in vitro studies [80]. In addition, the research conducted by Lirdprapamongkol et al. (2009) suggested that the presence of aldehyde and ketone in the structure of vanillin appeared to be responsible for its antimetastatic potential on A549 lung cancer cells by inhibiting hepatocyte growth factor (HGF)-induced phosphorylation of Akt on the one hand and deactivating phosphoinositide 3-kinase (PI3K) enzyme activity [81]. It can be hypothesized that the presence of aldehydes and ketones [TSLME: 2-Cyclohepten-1-one ( Tropilene) (Table 2); TSFME: 3,3-Dimethyl-2-butanone (Table 3)] in the structure of several compounds present in each extract might have certainly acted in the same way as the compound vanillin [81].

The morphological changes observed on the CBD, FD of MDA-MB-231 cells are probably due to synergistic or singular actions of the diversity of bioactive compounds such as flavonoids, terpenoids, and phenolic acids present in each part of the plant extract (Tables 2 and 3) [82, 83]. Moreover, the different morphological events that were observed on MDA-MD-231 cells through an inverted microscope and characterized by a decrease in the cell surface area (Fig. 9A: Black and yellow arrows), the appearance of plasma membrane blebs (Fig. 8A: Black arrows), and the condensation of the nucleus (Fig. 9A: Red arrows) are all some pieces of evidence as described by Hacker [84] to claim that TSLME and TSFME induced apoptosis in MDA-MB-231 cells. The induction of apoptosis on highly metastatic MDA-MB-231 cells [at 800 µg/mL (A1/B1)] can be attributed to the presence of compound vanillin in both extracts where it was previously demonstrated that incubation with vanillin led to higher apoptosis rate in highly metastatic breast cancer cell line MDA-MB-231 compared to the lowly metastatic MCF-7 breast cancer cells via induction through mitochondria-mediated signalling pathway through up-regulation of Bad and Bax proteins (pro-apoptotic) and inhibition of Bcl-2 protein functions Bcl-xl 5 (anti-apoptotic) which resulted in high expression of caspase-3, cytochrome-c, caspase-8 and cleaved PARP [Poly (ADP-ribose) polymerase)] along with more significant formation of apoptosis bodies in MDA-MB-231 than MCF-7 cell lines [85].

Conclusion

This work is the first study done on the cytotoxicity, antiproliferative, antimetastatic activities, and morphometric effects of Tecoma stans leaf and flower extracts on MDA-MB-231 triple-negative cells. Bioactive compounds found in Tecoma stans leaves methanolic extract (TSLME) and Tecoma stans flower methanolic extract (TSFME) are responsible for their cytotoxic, apoptotic, antiproliferative, and anti-metastatic effect on the MDA-MB-231 breast cancer cell lines are both concentration and treatment time-dependent. Therefore, on the one hand, the mechanisms involved in MDA-MB-231 cell apoptosis by TSLME and TSFME should be more elucidated. On the other hand, the mechanisms of action of the bioactive compounds detected in the methanolic extracts of Tecoma stans responsible for the antitumor activity of MDA-M-231 cells should be carried out for a much better understanding of their different modes of action which will contribute to a better optimal selection of therapeutic concentrations that can be used in chemotherapy without or lesser side effects than conventional pharmaceuticals.

Abbreviations

% yield

Percentage yield

AB

Antibacterial

AC

Anticancer

AFG

Antifungal

AFL

Antifouling

AHT

Anthelmintic

AIF

Anti-inflammatory

AM

Antimicrobial

AMR

Antimalarial

AO

Antioxidant

AP

Antiproliferative

ART

Antirheumatic

ASP

Antispasmodic

AST

Antiseptic

AT

Antitumor

BRC

Biotechnology Research Centre

CBD

Cell Body Diameter

CIU

Cyprus International University

CO2

Carbon dioxide

CP

Chemopreventive

CT

Cytotoxicity

DMEM

Dulbecco’s Modified Eagle Medium

DMSO

Dimethyl sulfoxide

ER

Estrogen Receptor

FBS

Fetal Bovine Serum

FD

Field Diameter

GC-MS

Gas chromatography-Mass Spectrophotometry

HER2

Human Epidermal Growth Factor Receptor 2

HP

Hepatoprotective

IMD

Immunomodulatory

MCF-7

Michigan Cancer Foundation-7

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAR

No Activity Reported

NRP

Neuroprotective

PBS

Phosphate-Buffered Saline

PR

Progesterone Receptor

PT

Process Thickness

TNBC

Triple Negative Breast Cancer

TSFME

Tecoma stans Flowers methanolic extract

TSLME

Tecoma stans Leave methanolic extract

WHO

World Health Organization

WHP

Wound Healing Potential

Authors’ contributions

SWTM, NR, OI: Methodology, Conceptualization, Project administration, Investigation, Writing–original draft, Writing—review & editing. NR: Supervision. All the authors listed in this paper have read and approved the final version of the submitted manuscript, and agreed to be accountable for the content of the work.

Funding

No external funding was received for this study.

Data availability

The datasets used and analysed during the present study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.