Phytochemical analysis and anticancer activity of the Pithecellobium dulce seed extract in colorectal cancer cells

: Colorectal cancer remains a challenging medical issue worldwide, and utilizing natural products and plants to produce novel, e ﬀ ective and safe therapies against this disease is continuously a sought-after strategy. Fruit and leaf extracts of Pithecellobium dulce ( P. dulce ) showed potential anticancer properties as they induced apoptosis of breast cancer and Dalton ’ s lymphoma ascites cells. Thus, the main objective of the current study is to determine whether the seed extract of P. dulce will a ﬀ ect apoptosis, cell cycle, migration, and in ﬂ ammation of LoVo colorectal cancer cells. The high-resolution liquid chromatography – mass spectrometry was used to determine the chemical composition of the P. dulce seed extract, which revealed the presence of 35 phytochemicals. The ﬁ ndings of this study indicated a signi ﬁ cant cytotoxic e ﬀ ect of seeds of this plant in colorectal cancer characterized by induction of apoptosis, cell cycle arrest, and reduction of migration. In addition, the seed extract suppressed several genes that are essential for cancer progression such as MMP2, MMP9, and IL-8, and, on the other hand, upregulated pro-apoptotic genes such as BAX and P53. This study has established P. dulce as a potential and valuable source for providing future therapies against colorectal cancer and other


Introduction
According to the Centers for Disease Control and Prevention, colorectal cancer is the third most common type of cancer and the third cause of cancer-related deaths in the United States.In 2019, the incidence rate of new colorectal cancer cases per 100,000 people in the United States was 36, of which there were 13 related deaths [1].These data emphasize the persistent necessity of seeking novel agents and compounds that can aid in the fight against colorectal cancer.
Plants and natural products have always been valuable sources of pharmacological agents that have historically been used in the treatment of a variety of human diseases [2][3][4][5][6].A prime example of these diseases is cancer as many pharmacological agents that are used against different types of cancer originated from plants [7].Vincristine, etoposide, irinotecan, and paclitaxel are a few examples of many anticancer drugs that work via different molecular mechanisms but all of them originate from plants [8][9][10][11].Irinotecan, which is a plant alkaloid, is one of the chemotherapeutic agents that is used to treat metastatic colorectal cancer with resistance to 5-fluorouracil [12].
Pithecellobium dulce (P.dulce) is a tree that can grow up to approximately 15 m in length, has a spiny trunk, and is a member of a flowering plant family called Fabaceae.This plant is native to areas such as South and Central America and India where it is traditionally used for edible and sometimes medicinal purposes [13].The medicinal purposes of P. dulce are due to the fact that it contains several ingredients known to improve human health.For example, seeds of P. dulce contain antioxidants such as flavonoids, quercetin, and vitamin C as well as several minerals, including calcium, magnesium, and phosphorous [14,15].Therefore, unsurprisingly, it was shown that this plant has beneficial uses in boosting immune function, strengthening bones and muscles, and managing symptoms of diseases such as dysentery and diabetes [16].It was reported that the P. dulce leaf extract induced apoptosis of breast cancer cell lines [17].Similar observations were found in Dalton's lymphoma ascites (DLA) cells as the fruit extract of P. dulce caused apoptosis of these cancer cells both in vitro and in vivo [18].
Although the role of P. dulce in colorectal cancer is not explored yet, there is sufficient evidence that this type of cancer is responsive to treatment derived from plants like Nux vomica [19], Vitis vinifera, Glycine max, and others [20].Therefore, this study aims to investigate the role of P. dulce in colorectal cancer.Specifically, the main objective of this project is to determine the phytochemical composition of P. dulce seeds and assess the cytotoxicity of the P. dulce seed extract against colorectal cancer cells.Another objective of this study is to determine the effect of the P. dulce seed extract on migration and inflammation in colorectal cancer cells.Completing the objectives of this study will potentially assist in developing new therapeutic agents of plant origin to treat colorectal cancer.

Plant collection and authentication
Fresh seeds of P. dulce were collected in May 2020 from Alaflaj province (22.2898579, 46.7188421) in the Riyadh region, Kingdom of Saudi Arabia.The seeds were botanically identified and authenticated by taxonomists at the herbarium of the Botany and Microbiology Department, College of Sciences, King Saud University.A voucher specimen of the seeds was deposited at the Herbarium unit in the College of Sciences, King Saud University (voucher number KSU No. 24593).

Plant extraction
Seeds of P. dulce were prewashed with distilled water to remove any contaminants and dried in darkness at room temperature for 15 days.The extraction was performed as previously described [21] in which 426 g of dried seeds were milled into a coarse powder using an electrical grinding mill.The coarse powder was then extracted three times by cold maceration in 80% methanol (Sigma-Aldrich, MO, USA) and incubated at room temperature for three days with intermittent shaking to prepare the crude extract.Following extraction, the crude extract was filtered using Whatman filter paper, grade 1 (Wagtech International Ltd, England).The filtrate was then dried using a rotary vacuum evaporator (Buchi Rotavapor R-210, Merck, Darmstadt, Germany) at 45 rpm and 40°C under controlled pressure and then stored in a refrigerator.

High-resolution liquid chromatography-mass spectrometry analysis
About 60 mg of the dried extract was extracted using MeOH/Acn/dH 2 O (40:40:20 v/v/v) into small molecules.The high-resolution liquid chromatography-mass spectrometry analysis was carried out using the Waters Acquity UPLC system coupled with a Xevo G2-S QTOF mass spectrometer equipped with an electrospray ionization source (ESI) (Waters Ltd., Elstree, UK) to identify the phytoconstituents of the P. dulce seed extract.The extracted metabolites were then introduced to an ACQUITY UPLC using an XSelect column (100 mm × 2.

Metabolites identification
Progenesis QI software (Waters Technologies, Massachusetts, USA) was used to detect peak selection and alignment of detected ions (m/z, Rt).Data were then processed by log transformation, mean centering, and Pareto scaling for univariate analyses.The identified metabolites were putatively annotated based on the following criteria: exact mass, fragmentation pattern, and isotopic distribution search against various databases including the PlantCyc database https://plantcyc.org/, Planta Piloto de Química Fina, Universidad de Alcalá database, NIST database, NIST spectra, and NIST Chemistry WebBook.

Cell viability analysis
Cell viability of LoVo, HUVECs, MCF7, and A549 treated with different concentrations of the P. dulce seeds extract was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Additionally, LoVo cells were treated with various concentrations of doxorubicin (MedChemExpress, NJ, USA), which was used as a positive control to compare its cytotoxicity with that of the P. dulce extract.Cells at a density of 1 × 10 4 cells were seeded on a 96well plate and cultured for 48 h at a 100 µL cell culture medium.Cells were then treated with five different concentrations of the extract (3.125, 6.25, 12.5, and 25 µg/mL) for 24 h.Following the incubation, MTT dissolved in 1× Dulbecco's phosphate-buffered saline (PBS) was added to each well and incubated for 3 h at 37°C and 5% CO 2 in darkness.After that, the culture medium containing MTT was discarded, and 100 µL of isopropranolol was added to each well to dissolve the purple crystals of formazan.The absorbance at 570 nm for each well was read using a microplate reader (BioTek, Elx-800, Agilent, CA, USA).Dose-response curves for each cell line were plotted, and OriginPro8.5 software was used to generate the half-maximal inhibitory concentration (IC 50 ), which was used in all subsequent experiments.

Annexin V-FITC/PI apoptosis detection
A FITC Annexin V apoptosis detection kit with propidium iodide (Biolegend, CA, USA) was used to evaluate the total cell death of LoVo cells.About 10 × 10 4 cells were seeded on 6-well plates overnight and then treated for 24 h.Cells were then harvested and washed twice with ice-cold 1× PBS and then resuspended in 100 µL of Annexin V binding buffer.After that, cells were stained with 5 µL of FITC Annexin V and 5 µL of propidium iodide for 15 min in darkness followed by the addition of 400 µL of Annexin V binding buffer.The stained samples were analyzed using a FACS flow cytometer (Cytomics FC; Beckman Coulter, USA).

Cell cycle analysis
Cell cycle analysis was carried out according to the manufacturer's protocol.Briefly, LoVo cells were seeded on 6well plates at a density of 1 × 10 5 and incubated overnight followed by treatment for 24 h.Following incubation, cells were collected and washed two times with an ice-cold 1× PBS and then fixed in 70% ethanol for 4 h at 4°C.After fixation, ethanol was discarded and cells were stained with propidium iodide at a concentration of 50 μg/mL for 30 min in darkness.Samples were then analyzed to assess cell cycle stages using a FACS flow cytometer (Cytomics FC; Beckman Coulter, USA).

Scratch assay
The migration of LoVo cells was assessed using the scratch assay.LoVo cells were cultured on 6-well plates until they reached 80% confluence.Then, a straight-line scratch was created using a sterile 200 μL pipette tip.Cells were then treated for 24 h, and images for the scratch were captured at 0-and 24 h post-scratch using a 4× objective of an EVOS XL Core microscope (Thermofisher, USA).The migration percent was determined by counting the number of migrated cells in the scratched area relative to untreated cells using ImageJ software (NIH Image, Bethesda, MD, USA).

Quantitative real-time polymerase chain reaction (qPCR)
Total RNA was extracted from LoVo cells treated for 24 h using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol.Reverse Transcription Master Mix for qPCR (MedChemExpress, NJ, USA) was used to synthesize cDNA.qPCR was carried out using Low ROX SYBR Green qPCR Master Mix (MedChemExpress, New Jersey, USA).Reverse and forward human primers for BAX, P53, MMP2, MMP9, P21, IL-8, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Integrated DNA Technologies (Leuven, Belgium), and the sequences of the primers are listed in Table 1.GAPDH was used as a housekeeping gene, and the expression for the gene of interest was normalized to the expression of GAPDH.The relative mRNA expression and the fold difference of the gene expression were calculated using the 2 −ΔΔCT method.

Statistical analysis
The statistical analysis was performed using GraphPad Prism 9 Software.Student's paired t-test was used to determine the statistical difference between the experimental groups, and data were presented as mean ± SD.The value of P < 0.05 was considered statistically significant.

The effect of the P. dulce seed extract on the cell viability of LoVo cells
The cytotoxic effects of the P. dulce seed extract on the cell viability for LoVo and HUVEC were evaluated using an MTT assay.The extract reduced the cell viability of treated LoVo cells in a concentration-dependent manner, as indicated in Figure 1(a).Extract concentrations of 3.125, 6.25, 12.5, and 25 µg/mL reduced cell viability to 48, 25, 19, and 14%, respectively.Doxorubicin had more cytotoxicity against LoVo cells than the extract (Figure 1(b)).Additionally, the extract exerted a less cytotoxic effect on HUVEC compared to LoVo cells (Figure 1(c)).Based on these data, the IC 50 of the extract was determined to be 3.03 ± 0.1 µg/mL.Therefore, 3 µg/mL of the extract was used to conduct all subsequent experiments on LoVo cells to further elucidate the potency and efficacy of this extract.The corresponding IC 50 value of the extract against HUVEC was 6.24 ± 0.25 µg/mL.IC 50 values of doxorubicin against LoVo and HUVEC were 0.98 ± 0.04 and 2.25 ± 0.12, respectively (Table 4).

The P. dulce seed extract-induced apoptosis of LoVo cells
To further investigate the potential cytotoxicity of the extract, the apoptosis rate of LoVo cells was evaluated following treatment with the extract.The extract induced a significant increase in the rate of cell apoptosis in the treated cells (Figure 2(b)).The rates of early and late apoptosis for treated cells were 23 and 16% compared to untreated cells, respectively.The impact of the extract on the apoptosis-related genes P53 and BAX was further evaluated.Extract treatment upregulated the gene expression of P53 and BAX by approximately 2-and 10-fold, respectively (Figure 2(c and d)).

The impact of the P. dulce seeds extract on the cell cycle of LoVo cells
The impact of the extract on cell cycle progression was evaluated for LoVo cells.As shown in Figure 3(b), the treatment with the extract resulted in a significant increase in the cell number at the sub-G1 phase to 27% compared to 0.5% of untreated cells.The increase in the cell number at the sub-G1 phase of the treated cells was accompanied by a decrease in the proportion of cells in the G1, S, and G2-M phases of the cell cycle to 46, 16, and 11%, respectively.Furthermore, the expression of the cell cycle inhibitor P21 gene was significantly upregulated in treated cells as compared to untreated cells (Figure 3(c)).

The P. dulce seed extract suppressed the migration of LoVo cells
Scratch assay was used to examine the influence of the extract on cell migration during wound closure of LoVo cells.The results showed a clear decrease in the migration rate of LoVo cells following extract treatment.The quantification of migrated cells revealed that the treatment caused a 78% reduction in the migration rate (Figure 4(b)).The reduction in cell migration was accompanied by a significant downregulation in the gene expression of MMP2 and MMP9 in the treated cells compared to untreated cells (Figure 4(c and d)).

The P. dulce seed extract modulated the gene expression of inflammatory cytokines in LoVo cells
The next step was to investigate the effect of the P. dulce seed extract on inflammatory signaling as it has been well-established that inflammation plays a crucial role in cancer progression and treatment.To assess the impact of the extract treatment on inflammation, LoVo cells were  treated and then the gene expression of two inflammatory cytokines IL-8 and TNF-α was measured.As shown in Figure 5(a), the extract significantly reduced the relative gene expression of IL-8 as compared to untreated cells.Conversely, the TNF-α gene expression was significantly upregulated in the extract-treated cells as compared to the control (Figure 5(b)).

Exploring the effect of the P. dulce seed extract on cell viability of breast and lung cancer cells
Since several genes that were impacted by P. dulce seeds play roles that are not exclusive to colorectal cancer and can expand to other cancers such as lung and breast cancers, this study opted to determine whether the extract will also affect these cancers.To achieve this aim, MCF7 and A549 cells were treated with various concentrations of the extract to assess their impact on cell viability.The extract reduced the cell viability of both MCF7 and A549 cells in a concentration-dependent manner (Figure 6).

Discussion
Utilizing plants and natural products to produce effective therapies against different types of cancer has proven to be a successful strategy in the past [7].Fruits and leaves of P. dulce were shown to exert cytotoxic effects in some types of cancer [17,18].However, the anticancer activity of P. dulce has not been explored in colorectal cancer.Additionally, it remains unknown whether other parts of the P. dulce plant such as seeds will show anticancer activity similar to fruits and leaves.Therefore, this study aimed to further expand the investigation of the role of P. dulce by testing the effect of its seeds on cell viability, apoptosis, migration, and inflammation of colorectal cancer cells.Identification of phytochemical constituents of the P. dulce seed extract would be necessary to detect phytoconstituents with known anticancer properties and to correlate them with the observed biological activities.Our results revealed the presence of 35 phytochemicals in the methanolic extract of the P. dulce seeds, five of them with known anticancer properties, including octadecanedioic acid, HAS, linoelaidic acid, soyasaponin III, and kaempferol 7-O-beta-D-glucopyranoside.HAS is a long-chain, naturally occurring fatty acid and contains various isomers of HAS.However, in this study, we were not able to differentiate between different isomers of HAS as they have equal molecular masses.Several regioisomers of HAS have been studied for their cytotoxic effects against various types of cancer cells.HAS derivatives such as 5-HAS, 7-HAS, and 9-HAS showed antiproliferative effects against colorectal, prostate, and breast cancer cells [25].Additionally, in colorectal adenocarcinoma cells (HT29), other studies showed that HAS-9 induced cell cycle arrest G0/G1 via upregulating P21 expression and cell differentiation via inhibition of histone deacetylase 1 [24,27].Octadecanedioic acid, an, α,ω-dicarboxylic acid, is a natural product that has been found in Pinus radiata and Arabidopsis thaliana.A study reported an improvement in the efficacy of paclitaxel conjugated with octadecanedioic acid as compared to two FDA-approved paclitaxel formulations against the subcutaneous murine xenograft model of human colorectal adenocarcinoma and pancreas ductal carcinoma [26].
The other identified phytochemical, linoleic acid, was found to inhibit the proliferation of colorectal cancer cells.This effect was attributed to the ability of linoleic acid to induce oxidative stress and mitochondrial dysfunction [23].Another study revealed that linoleic acid suppressed growth and induced apoptosis of gastric and colon cancer cells in a dose-dependent manner along with a decrease in cancer cell invasion in vitro and metastatic foci in the peritoneal cavity in vivo [28].Soyasaponin III has been shown to inhibit the growth of colorectal adenocarcinoma cells and reduce the activity of protein kinase C activity [29].Another study reported the proliferation and inhibition of hepatocarcinoma Hep-G1 cells by an extract containing 62% soyasaponin I and 29% soyasaponin III [30].The other phytochemical identified in the P. dulce seed extract, kaempferol 7-O-beta-D-glucopyranoside, has been shown to exert an antiproliferative effect and induce apoptosis and cell cycle arrest at the G2/M phase against HeLa cervical cancer cells.Indeed, these changes were also associated with the inhibition of NF-κB translocation, upregulation of BAX, downregulation of BCL-2, cyclin B1, and cdk1 [22].Overall, these data indicated the anticancer activity of these phytochemicals and support the findings of our study about the potential anticancer effect of P. dulce seeds.Additionally, the anticancer activity of other identified phytochemicals has not been tested before and it would be interesting to evaluate their potential cytotoxicity against cancer cells.
The results of this study demonstrated the ability of the P. dulce seed extract to reduce the viability of colorectal cancer cells along with the induction of apoptosis.Furthermore, the extract induced upregulation of BAX and P53, which are known mediators of cell apoptosis.Therefore, these data further supported the assertion of the cytotoxicity of P. dulce seeds.These data are in line with a previous study that showed the potent and selective cytotoxicity of the P. dulce leas extract against breast cancer cells [17].Dhanisha et al. found that the P. dulce fruit extract had both in vitro and in vivo cytotoxicity against DLA cells [18].Thus, the results of this study indicated that like the effect of P. dulce fruits and leaves on breast cancer and DLA cells, the seeds of this plant also induced cytotoxicity in colorectal cancer cells.
While other studies focused mainly on the apoptotic effect of P. dulce, this study aimed to expand the investigation to include the effect of this plant on cell cycle and  migration.This study demonstrated a substantial elevation in the cell population in the sub-G1 phase; the cells in this phase are recognized by reduced DNA content as a result of DNA fragmentation [31].The reduction in DNA content is a typical feature of apoptotic cells but it is not clear whether the observed increase in cells at the sub-G1 phase was attributed to the increase in cell apoptosis or was a result of cell cycle arrest of viable cells at the sub-G1 phase.The P21 gene is one of the main regulators of the cell cycle and inhibits cyclin-dependent kinase, thereby arresting the cell cycle and inhibiting cell division [32].The P. dulce seed extract upregulated the expression of the P21 gene, which might contribute to the effect observed on the cell cycle.
In addition to determining the effect of the P. dulce seed extract on apoptosis and cell cycle progression of colorectal cancer cells, this study also investigated the influence of this plant on the migration of these cells.The findings of the current study revealed that the P. dulce seed extract significantly inhibited the migration of colorectal cancer cells.This result is noteworthy because, to the best of our knowledge, our study is the first to demonstrate the effect of the P. dulce seed extract on the migration of cancer cells.Furthermore, the P. dulce seed extract-induced suppression of migration was accompanied by the downregulation of MMP2 and MMP9 genes.These data would suggest a potential role of the P. dulce seed extract in mitigating colorectal cancer progression since MMP2 and MMP9 have been shown to be critical mediators for cancer migration, invasion, metastasis, and progression.
It is well-established that inflammatory signaling plays a major role in cancer pathophysiology where several inflammatory cytokines such as IL-8 and TNF-α can influence the progression, migration, and apoptosis of different types of cancer, including colorectal cancer [33].Evidence from a previous study showed that the fruit extract of P. dulce had anti-inflammatory effects, displayed by reducing the expression of multiple cytokines such as IL-1ß and IL-6 [34].In this study, the P. dulce seed extract suppressed the gene expression of IL-8 in colorectal cancer cells, which concurs with other studies demonstrating an anti-inflammatory role of this plant [34,35].However, the P. dulce seed extract had the opposite effect on TNF-α as it increased the gene expression of this cytokine.Notably, TNF-α has complex multifaceted roles in cancer pathophysiology where it can induce apoptosis of cancer cells in some cases and promote cancer in others [36].Therefore, there is a possibility of a dual action of P. dulce where it suppresses the expression of IL-8, thereby contributing to the inhibition of cancer progression, while enhancing the expression of TNF-α, thereby inducing the apoptosis of cancer cells.
Even though the main focus of this study was to evaluate the role of the P. dulce seed extract in colorectal cancer, further investigation was conducted to assess whether the anti-cancer effect of this seed extract is exclusive to colorectal cancer cells or whether it can extend to other cancer.Our findings indicated that while the effect on colorectal cancer cells appeared to be more potent, the P. dulce seed extract induced a reduction in the cell viability of both breast and lung cancer cells.Thus, the seed extract of this plant can have applications beyond its effects on colorectal cancer but further studies to delineate the exact roles of P. dulce seeds in other cancers are warranted.

Conclusion
The current study demonstrates the potential anticancer effect of P. dulce seeds on colorectal cancer cells.Indeed, this study further adds to the list of therapeutic benefits of this plant that were already reported, such as enhancing immune function, strengthening bones and muscles, and improving symptoms associated with dysentery and diabetes.Additionally, the findings of this study suggest that P. dulce could potentially be used in cancer therapy as many anticancer agents are developed from plants.Future studies should investigate the molecular mechanisms that mediate these anticancer effects of P. dulce seeds.There is also a need to conduct additional studies that can aid in defining the broader roles of this plant in other types of cancer.Finally, it will be interesting to gauge the value of using P. dulce seeds in combination with classic chemotherapeutic agents to enhance their efficacy and/or to lower their toxicities.

Figure 1 :
Figure 1: The cell viability of the extract-treated LoVo cells (a), doxorubicin-treated LoVo cells (b), and the extract-treated HUVEC (c).LoVo and HUVEC cells were treated with different concentrations of the extract ranging from 3.125 to 25 µg/mL to assess the impact of the extract on cell viability as measured by MTT assay.LoVo cells were also treated with different concentrations of doxorubicin (0.625-5 µg/mL) to compare their cytotoxicity with the extract cytotoxicity.Data were represented as a percentage of cell viability ± SD.

Figure 2 :
Figure 2: The apoptotic effect of the P. dulce seed extract on LoVo cells.(a) Representative apoptosis histograms for untreated cells and cells treated with the extract following staining with FITC Annexin V and propidium iodide.(b) Percentage of early apoptotic, late apoptotic, and necrotic cells following treatment with the extract as compared to untreated cells.(c) The expression of apoptosis-related gene P53 in treated cells compared to untreated cells.(d) The expression of apoptosis-related gene BAX in treated cells compared to untreated cells.Data were represented as the mean ± SD and P < 0.05 was considered statistically significant.

Figure 3 :
Figure 3: The impact of the P. dulce seed extract on the cell cycle of LoVo cells.(a) Representative images of cell cycle analysis for untreated cells and cells treated with the extract following staining with propidium iodide.(b) Cell population percentage in cell cycle phases: sub-G1, G1, S, and G2-M in treated cells compared to untreated cells.(c) The gene expression of the cell cycle inhibitor P21 in treated cells compared to untreated cells.Data were represented as the mean ± SD and P < 0.05 was considered statistically significant.

Figure 4 :
Figure 4: The effect of the P. dulce seeds extract on cell migration in LoVo cells.(a) Representative images of cell migration taken at the beginning of the experiment (0 h) and 24 h post-scratch using 4× objective for untreated cells and cells treated with the extract.(b) Percentage of cell migration rate of cells following treatment with the extract relative to untreated cells.(c) The gene expression of MMP2 following treatment with the extract relative to untreated cells.(d) The gene expression of MMP9 following treatment with the extract relative to untreated cells.Data were represented as the mean ± SD and P < 0.05 was considered statistically significant.

Figure 5 :
Figure 5: The effect of the P. dulce seed extract on inflammation in LoVo cells.(a) The IL-8 gene expression in the extract-treated cells compared to untreated cells.(b) The TNF-α gene expression in the extract-treated cells compared to untreated cells.Data were represented as the mean ± SD and P < 0.05 was considered statistically significant.

Figure 6 :
Figure 6: The effect of the P. dulce seed extract on the viability of MCF7 and A549 cells.Cells were exposed to different concentrations of the extract ranging from 3.125 to 25 µg/mL to assess the impact of the extract on cell viability as measured by the MTT assay.Data were represented as a percentage of cell survival ± SD.

Table 1 :
Forward and reverse primer sequences

Table 2 :
Identified compounds of the P. dulce seed extract on the positive electrospray ionization modes

Table 3 :
Identified compounds of the P. dulce seed extract on the negative electrospray ionization modes

Table 4 :
The corresponding IC 50 values of the P. dulce seed extract and doxorubicin against LoVo and HUVEC cells