Dual antineoplastic and photodynamic effects of methanolic extract of Tecoma stan s yellow flowers for cancer treatment

Tecoma stans methanolic flower extract (TSFE) contained coumarins and anthocyanins that may act as photosensitizers. The TSFE showed cytotoxicity against lung cancer and breast cancer cell lines after being irradiated. Therefore, TSFE may be a promising natural photosensitizer to be used in photodynamic therapy of different cancers.


Introduction
Plants have been used for centuries as the main source of medication in folk medicine, especially in developing countries.They are considered cheaper and safer with fewer adverse reactions than many synthetic chemicals.Moreover, plants contain several phytochemical constituents which possess a wide range of biological activities (1).
Tecoma stans (L.) Juss.Ex Kunth (Bignoniaceae) is a fastgrowing ornamental plant grown in Egypt and in many tropical and subtropical areas (2,3).It is widely used in folk medicine for several purposes.Previous pharmacological studies have revealed that T. stans has various biological activities such as antidiabetic, anticancer, antioxidant, antispasmodic, antimicrobial, and antifungal activities (4)(5)(6)(7).The wide range of activities is attributed to a diversity of phytoconstituents isolated from different parts of the plant.
In the field of cancer treatment, searching for anticancer drugs that are derived from plants has attracted great attention.The anti-cancer plant-derived compounds may be direct cytotoxic compounds or photosensitive ones called photosensitizers.A photosensitizing (PS) agent can be excited with a specific wavelength of light in the presence of tissue oxygen.This photochemical interaction results in the generation of reactive oxygen species and free radicals that are cytotoxic to the cells.These cytotoxic species exert their cytotoxic effects only at the site where they are generated.Therefore, by exposing the site of action only to the specific light wavelength, a high degree of selectivity can be attained.This light-dependent modality of treatment is known as photodynamic therapy (PDT) (4,8), which is considered a promising choice for cancer therapy because of its low systemic toxicity and can be used repeatedly.
A significant challenge in the field of PDT is to get photosensitizers from natural plant extracts.Natural photosensitizers are preferred to synthetic ones owing to their high production yield, high biodegradability, high safety, efficacy, and low cost (9,10).It has been reported that herbal remedies that include a mixture of PS phytochemicals can be employed as alternatives to conventional PSs for PDT (11,12).The chemical structures of several phytochemicals contain chromophores that can absorb light at appropriate wavelengths effectively.Thus, these phytochemicals are effective PDT candidates but need to be investigated for their chemical characteristics, provenance from nature, and photo-pharmaceutical characteristics (12).
In this context, T. stans is one of the promising plants that may contain PS agents, which can be used in PDT, in addition to other cytotoxic phytoconstituents.
This study aims to determine whether a methanolic extract of T. stans flowers (TSFE) can be utilized to treat cancer both conventionally and using PDT.Flowers were selected due to their high content of many phytocomponents with various activities.We analyzed the phytochemical components of TSFE and investigated both the cytotoxicity and the photodynamic activities of the whole extract on breast cancer and lung cancer cell lines.

Materials and Methods
Plant collection extract preparation Flowers of T. stans were collected from El merryland garden (Heliopolis, Cairo, Egypt) in August 2019.This plant material was identified and authenticated at the National Research Center, Egypt.Fresh flowers were collected, washed with distilled water, dried on a 3 mm PC sheet for one hour at room temperature, and cut into small pieces for further evaluation.
The small pieces of the fresh flowers (0.6 kg) were soaked in methanol and ground using an electric mixer then, transferred to a 5-L percolator and allowed to dropwise at a rate of 5 drops/min, methanol was added (500 mL) until complete exhaustion of the flower's marc.The collected methanol extract was evaporated under reduced pressure using a rotavapor (Heidolph, Germany) until complete dryness.The dried extract (138 gm equal to 23%w/w fresh flowers) was stored in a quick-fit flask in the dark and frozen for further phytochemical and biological analysis.The extract was prepared in dim laboratory light and kept in darkness.
Qualitative estimation of TSFE by ultra-performance liquid chromatography coupled to tandem high-definition mass spectrometry (UPLC-MS/MS) Sample preparation UPLC-MS/MS analysis was done, using AB Sciex TripleTOF 5600+, in the basic research department, proteomics unit, at 57357 Children's Cancer Hospital (Alsayeda Zeinab, Cairo, Egypt).
First, a reconstitution working solvent was prepared as a mixture of deionized water, methanol (purchased from Fisher Scientific, UK), and acetonitrile (purchased from Sigma-Aldrich, Germany) in ratios of 50: 25: 25, respectively.One milliliter of the prepared solvent was added to 50 mg of weighted TSFE and vortexed for 2 minutes followed by ultrasonication for 10 minutes and centrifugation for 10 minutes at 10 000 rpm.Then, 50 µL of the prepared extract solution was diluted with 1 ml of the reconstitution solvent.Finally, 10 µL of the extract solution (contained 1 µg/µL TSFE) was injected in positive mode and negative mode. 10 µL of the reconstitution solvent was injected as a blank sample.

Acquisition Method
Data processing MS-DIAL 3.52, Database was used: Respect negative (1573 records) and positive (2737).Master View was used for feature (peaks) extraction from Total ion chromatogram (TIC) based on the following criteria: Features should have a Signal-to-Noise greater than 5 (non-targeted analysis).Feature's intensities of the sample-to-blank should be greater than 3. Spectrophotometric analysis UV-visible absorption spectra of a diluted methanolic TSFE were recorded in the wavelength range of 200-800 nm using a Rayleigh 2601 double-beam spectrophotometer (Beijing, China), using methanol as a reference blank.

Cell culture
The cytotoxicity of TSFE was tested using two cell lines, Breast Adenocarcinoma cells (MCF-7) and Lung Cancer cells (A-549).Both cell lines were obtained from Nawah Scientific Inc., (Mokatam, Cairo, Egypt).Cells were cultured in DMEM media supplemented with 100 mg/ mL of streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated fetal bovine serum (all obtained from Lonza, Belgium), and incubated in humidified 5% (v/v) CO2 atmosphere at 37°C.

Dark and photo-cytotoxicity
Aliquots of 100 μL cell suspension (5×10 3 cells) were seeded in 96-well plates and incubated in complete media for 24 hours.Cells were treated with another aliquot of 100 μL media containing the flower extract at various concentrations (0.01 to 100 μg/L) and incubated for 72 hours.For dark cytotoxicity, the cell viability was assessed by sulforhodamine B (SRB) assay, immediately after the incubation period.The media was replaced with 150 μL of 10% trichloroacetic acid (TCA) to fix the cells and incubated at 4°C for 1 hour.The TCA solution was removed and the cells were washed 5 times with distilled water.Aliquots of 70 μL SRB solution (0.4% w/v) were added and incubated in a dark place at room temperature for 10 min.Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight.Then, 150 μL of tris(hydroxymethyl)aminomethane (10 mM) was added to dissolve the protein-bound SRB stain; the absorbance was measured at 540 nm using a BMG LABTECH®-FLUOstar Omega microplate reader (Ortenberg, Germany).
For photocytotoxicity determination, the cells were irradiated after the 72 hours incubation period.The irradiation was done using a light emitting diode (LED) light source fitted with a 450 nm band path filter (Photon scientific, Cairo, Egypt) at 90 mW/cm 2 for 15 minutes.Afterward, an SRB assay was conducted as described above.

Results
Identification of the compounds Interpretation of UPLC/MS/MS was conducted using the database of 57357 Children's Cancer Hospital library (Alsayeda Zeinab, Cairo, Egypt).The spectra of the unknown components were compared to the spectra of the known authentic reference standards from the library.Eighty-seven and 44 compounds were identified from the positive acquisition mode (Figure 1) and the negative acquisition mode (Figure 2), respectively.

Spectrophotometric analysis
The UV-VIS spectrum of the diluted methanolic TSFE (Figure 4) showed a wide absorption spectrum in the UVA and visible regions with characteristic peaks at the range of 400-450 nm.
Cytotoxicity assay As illustrated in Figure 5, TSFE showed negligible cytotoxicity against McF-7 cell lines at all the tested concentrations in dark.Upon radiation at 450 nm, the cell viability was 84% at the highest tested concentration (100 μg/mL), but the decrease was statistically non-significant (P > 0.05).On the other hand, the extract showed significant dark and photo-cytotoxicity on A549 cell lines at higher concentrations (Figure 6).At a concentration of 100 μg/mL, the cell viability in dark was 78%, which significantly decreased to 64% after irradiation (P < 0.05).Wavelength (nm)

Absorbance
These results suggest that TSFE is a promising natural photosensitizer for PDT.

Discussion
In this study, we analyzed the phytochemical components of TSFE by UPLC/MS.Each of the identified compounds has a wide range of biological activities as reported by several previous studies, as summarized in Supplementary file 1 (Table S1).Therefore, TSFE can be considered a rich source of natural bioactive constituents that may exert different biological activities.TSFE has previously been studied for different biological activities.Ha et al isolated five components from TSFE and proved their activities as alpha-glucosidase inhibitors (5).Sugavanam et al reported CNS depressant activity of the TSFE (34).Gonçalves et al identified the phytoconstituents of several classes of TSFE and studied their antibacterial activity (35).Here, we focused on the antineoplastic activity of TSFE, either due to the presence of cytotoxic components, PS agents, or both.For this purpose, the cytotoxicity of TSFE was evaluated on two different cancer cell lines.The assessment of cytotoxicity was carried out in dark and after blue light irradiation to assess the photodynamic activity of TSFE.The results showed significant cytotoxicity of TSFE against the A-549 lung cancer cell line.These are consistent with those obtained by Robinson et al who reported high cytotoxicity of methanolic TSFE on A-549 cells (36).This high cytotoxicity may be attributed to the presence of phytoconstituents that exert antineoplastic activities, as revealed by UPLC/MS results (Table 1).Examples of identified constituents with proposed antineoplastic activity are apigenin (37), rosmarinic acid (38), esculin (39), genistein (40), luteolin (41), kaempferol (42), and isorhamnetin (43).
Moreover, our results revealed that the cytotoxicity was enhanced significantly after light radiation.The UV-visible spectrum of TSFE (Figure 4) showed a strong absorption peak in the visible region, especially in 400-450 nm, suggesting that TSFE could be excited by visible blue light, thus it might be a promising photosensitizer (44).The high photo-cytotoxicity may be due to the presence of PS phytoconstituents such as coumarins (as esculin and daphnetin) and anthocyanins (revealed from the results of UPLC/MS).These compounds can generate reactive oxygen species upon excitation by light.
Coumarins are organic dyes that have shown good PS properties in the treatment of many skin conditions such as psoriasis and vitiligo (12,45,46).In addition, anthocyanins derivatives, contained in TSFE, show photochemical and photophysical properties as they can absorb light in the blue wavelength (400-450 nm), which makes them potential PS in PDT of cancer (47).
A few previous studies have addressed the PDT activity of TSFE.Mamone et al reported that TSFE was a good photosensitizer in the photodynamic inhibition of the Staphylococcus epidermis (44).On the other hand, Tariq et al have studied the T. stans branch and leaves extract on the rhabdomyosarcoma cell line.They reported marked cytotoxicity of the tested extract, which was significantly enhanced upon the combination with a photosensitizer and silver nanoparticles due to the synergistic mechanism between anticancer and photodynamic activity (4).Our results showed that the cytotoxicity against breast cancer cell McF-7 was not significant in dark, but it increased non-significantly by applying the light that enhance the poor activity.
The fruit extract of T. stans was reported to be highly cytotoxic against the HEPG-2 cell line but exhibited lower cytotoxicity against the MCF-7 cancer cell line (2).However, Thirumal et al reported a good antiproliferative effect of ethanolic TSFE on MCF-7 cells with IC50 of 70 μg/mL (48).Moreover, the ethanolic bark extract of T. stans was able to induce apoptosis in human breast cancer cell lines at all concentrations (49).These studies used different parts of the plant and different types of extract that may contain different phytoconstituents with various concentrations.In this study, MCF-7 cells might be resistant to the components of TSFE as they were reported to develop resistance to many chemotherapeutic drugs (50).Slight enhancement of the cytotoxicity on MCF-7 after light radiation proves that the TSFE is a promising PS in PDT, but further investigation is needed.

Conclusion
The current investigation revealed that the methanolic extract of T. stans flowers contained a great number of bioactive constituents responsible for many biological activities.The phytochemicals identified through UPLC-MS analysis confirmed the presence of both antineoplastic phytoconstituents and PS compounds.This makes the flower extract of T. stans a promising anticancer drug and promising PS in PDT.This study could be a promising base for further investigations in other biological and medical aspects.

Authors' contribution
MF and NEA were responsible for conceptualizing and supervising the whole work.AYK and DAF were responsible for carrying out the experimental work and preparing the initial draft of the manuscript.All authors

Figure 1 .
Figure 1.Total ion chromatogram in positive ionization mode of methanolic extract of Tecoma stans yellow flowers.

Figure 2 .
Figure 2. Total ion chromatogram in negative ionization mode of methanolic extract of Tecoma stans yellow flowers.

Figure 3 .
Figure 3. Pie chart showing different phytochemical classes identified via UPLC/MS/MS of crude methanolic extract of Tecoma stans yellow flowers.

Figure 4 .
Figure 4.A UV-Visible spectrum of Tecoma stans flower extract.

Figure 5 .
Figure 5.In vitro cell viability of breast cancer cell lines (McF-7) when treated with different concentrations of Tecoma stans methanolic flower extract (TSFE) in the dark (A) and after light irradiation (B).

Figure 6 .
Figure 6.In vitro cell viability of lung cancer (A-549) cell lines when treated with different concentrations of Tecoma stans methanolic flower extract (TSFE) in the dark (A) and after light irradiation (B).

Table 1 .
UPLC/MS/MS of the phytochemical components of methanolic extract of Tecoma stans yellow flowers (TSFE)