Metabolite Profiling of Colvillea racemosa via UPLC-ESI-QTOF-MS Analysis in Correlation to the In Vitro Antioxidant and Cytotoxic Potential against A549 Non-Small Cell Lung Cancer Cell Line

In this study, flower and leaf extracts of Colvillea racemosa were considered a source of bioactive compounds. In this context, the objective of the study focused on investigating the anticancer potential as well as the phytochemical composition of both extracts. The extracts were analyzed by UPLC-ESI-QTOF-MS, and the bioactivity was tested using in vitro antioxidant assays (FRAP, DPPH, and ABTS) in addition to cytotoxic assays on non-small cell lung cancer cell line (A549). Our results clearly indicated the potent radical scavenging capacity of both extracts. Importantly, the flower extract exhibited a greater antioxidant capacity than the leaf extract. In terms of cytotoxic activity, leaf and flower extracts significantly inhibited cell viability with IC50 values of 17.0 and 17.2 µg/mL, respectively. The phytochemical characterization enabled the putative annotation of 42 metabolites, such as saccharides, phenolic acids, flavonoids, amino acids, and fatty acids. Among them, the flavonoid C-glycosides stand out due to their high relative abundance and previous reports on their anticancer bioactivity. For a better understanding of the bioactive mechanisms, four flavonoids (vitexin, kaempferol-3-O-rutinoside, luteolin, and isoorientin) were selected for molecular docking on hallmark protein targets in lung cancer as represented by γ-PI3K, EGFR, and CDK2 through in-silico studies. In these models, kaempferol-3-O-rutinoside and vitexin had the highest binding scores on γ-PI3K and CDK2, followed by isoorientin, so they could be highly responsible for the bioactive properties of C. racemosa extracts.


Introduction
Family Fabaceae is a valuable source of many phytochemicals, viz., phenolic acids, flavonoids, saponins, and alkaloids with an anti-cancer potential [1].Importantly, polyphenolic compounds are primary competitors that can reverse carcinogenesis through inhibi- tion of oxidative stress, induction of cell cycle apoptosis, and suppression of the signaling molecules proliferation [2].Many studies reported the antioxidant activity of different plants in this family.For instance, Go devac et al. [3] studied nine Fabaceae species growing in Serbia and Montenegro, where Lathyrus binatus, Trifolium pannonicum, and Anthyllis aurea revealed as the most potent antioxidant plants using different antioxidant assays.Additionally, the seed extract of Mucuna pruriens provoked antioxidant effects using DPPH, FRAP, and ABTS assays.The results were comparable to those of rutin and gallic acid used as standards [4].Moreover, the family Fabaceae has been studied for cytotoxic potential.An alkaloidal fraction prepared from Indigofera suffruticosa (a member of the family Fabaceae) exhibited cytotoxic activity with IC50 values of 37.39 and 24.13 µg/mL against LM2 (breast adenocarcinoma) and LP07 (lung adenocarcinoma) cell lines, respectively [5].Also, the extract of Senna singueana at 100 mg/kg showed an in vivo antiapoptotic effect on liver cells by reduction of Bcl-2 [6].Importantly, other studies revealed low cytotoxicity of other plants belonging to Fabaceae on the Vero cell line [7].
Colvillea racemosa Bojer ex Hook, also known as the whip tree, is an exotic tree that belongs to the Fabaceae family [8].It represents the only species of the genus Colvillea, which is why it is described as a monotypic plant [8].It is the most beautiful deciduous tree native to the western coast of Madagascar.However, it is widely cultivated as an ornamental plant in some tropical countries such as Australia and North America [8,9].It is characterized by beautiful, inspiring clusters of bright orange flowers, so it is commonly known as Colville's Glory.Rivers et al. (2010) [10] reported the morphological similarity between Colvillea racemosa and Delonix regia or royal poinciana, which are endangered Malagasy species.However, Colvillea had a unique genetic material with a greater number of special alleles.In fact, it was placed on the red list as a threatened species according to the International Conservation of Nature and Natural Resources (IUCN).
Morphologically, the leaves of C. racemosa are small compound, bipinnate leaflets with a deep green color [11].Scientific studies have shown that leaf extracts exhibit multiple pharmacological activities, such as antioxidant, cytotoxic, and antimicrobial activities, in addition to their antineoplastic activity against Ehrlich carcinoma [9,11].These bioactive properties are associated with the phytochemical composition of this matrix, which has been characterized mainly by the presence of phenolic compounds (e.g., hesperidin, naringin, vanillic and benzoic acids) [11] and dihydrochalcones [8].However, only a very small number of studies have focused on the phytochemical characterization of C. racemosa extracts.Therefore, there is a need to explore in detail the phytochemical compounds present in this species, which can help to better understand the bioactive properties described.
Furthermore, several classes of chemical compounds, such as flavonoids, diterpenes, and steroids, have been isolated from the closely related genus Caesalpinia [12].Members of this genus are endowed with an unusual combination of biologically active carotenoids and anthocyanins, particularly cyanidin-3-O-rutinoside, which holds great promise in food and pharmaceutical applications [13].
Lung carcinoma remains the first leading cause of death worldwide and has a low survival rate, according to recent reports from the World Health Organization.In 2020, it was responsible for more than one million deaths, accounting for approximately 18% of all cancer deaths [14].Lung cancer can be divided into small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) groups [15].NSCLC constitutes approximately 85% of all lung cancer cases [16,17].Notably, surgery is the preferred and main approach for early-stage NSCLC, which offers a significant chance for long-term survival for those patients [18,19].However, late stages require platinum-based combinations as chemotherapeutics [20].
Chemotherapy and radiotherapy are the most common conventional therapies for cancer treatment.However, they suffer from drug resistance and serious side effects [21].Therefore, as a potential future alternative, plant-based secondary metabolites show promising anticancer activities with multiple targets.
Importantly, oxidative stress stimulates the signaling pathway phosphatidylinositol 3-kinase (PI3K)/AKT, which in turn triggers apoptosis [22].Accordingly, PI3K and other kinases have emerged as attractive targets for the therapeutic intervention of cancer.PI3K plays a crucial role in the DNA repair cell cycle, cellular metabolism, and programmed cell death [23].Similarly, the epidermal growth factor receptor (EGFR) is another type of kinase that plays a key role in cell growth, differentiation, and tumorigenesis.A previous study by Filosto et al. [24] demonstrated that oxidative stress induced aberrant phosphorylation of the EGFR in the A549 adenocarcinoma model.Therefore, targeting these two pathways is considered 0a promising therapeutic strategy for a variety of malignancies such as stomach, breast, colon, and lung [23,25].
Another key player in cancer pathogenesis is CDK2, which is a serine/threonine protein kinase that plays a role in the G1/S transition, the initiation of DNA synthesis, and the regulation of the exit from the S phase.CDK2 is altered in 0.16% of all cancers, yet specifically colon adenocarcinoma possesses the greatest prevalence of alterations [26].
Recently, there has been a growing interest in natural antioxidants that can capture reactive oxygen species (ROS) production, reduce oxidative damage, induce DNA damage, and decrease the generation of anti-apoptotic protein levels [27].In this context, there is still a great need to investigate the mechanisms of action of these plant-based secondary metabolites, which may lead to possible pharmacological alternatives for cancer treatment in the future.
To the best of our knowledge, only a few studies on Colvillea have been conducted.Therefore, our study aimed to comprehensively investigate the bioactive metabolites of C. racemosa leaf and flower extracts using UPLC-ESI-QTOF-MS and their antioxidant potential as well as their cytotoxic effects targeting A549 non-small cell lung carcinoma (NSCLC) by in-vitro assays and molecular modeling approaches.We anticipate that this study will provide guidelines for the development, utilization, and pharmaceutical application of C. racemosa.

Total Phenolic and Flavonoid Contents
As depicted in Table 1, both phenolic and flavonoid contents of the flower extract of C. racemosa were approximately three times higher than that of the leaf extract (98.24 and 86.68 versus 34.99 and 24.14, for total phenolics and flavonoids calculated as mg gallic acid/g extract and rutin equivalents/g extract, respectively).a, b, c, Different letters denote significant differences between treatments using # unpaired t-test and * one-way ANOVA followed by Tukey's at p < 0.05.

In-Vitro Antioxidant Assays
Radical scavenging capacity is essential for suppressing the harmful effects of free radicals on the human body.Leaf and flower extracts of C. racemosa were evaluated for their antioxidant potential using three different models of antioxidant assays, viz., FRAP, DPPH, and ABTS assays.The results clearly indicated the antioxidant activities of both extracts (Table 1).However, the flower extract exhibited a significantly higher antioxidant capacity (IC 50 : 190 ± 8.0 µM TE/mg extract) for reducing Fe 3+ to Fe 2+ in an acidic medium in the FRAP assay compared to the leaf extract (IC 50 : 362 ± 24 µM TE/mg extract).Similarly, the flower extract showed a more potent radical scavenging capacity (IC 50 : 177 ± 2.0, 26 ± 2.0 µg/mL) than the leaf extract (IC 50 : 381 ± 2.0, 36 ± 2.0 µg/mL) using DPPH and ABTS methods, respectively.The unique diversity of flavonoid structures is what ultimately results in a marked difference in biological activities.In fact, flavonoids (kaempferol, luteolin, and apigenin), as well as non-flavonoid compounds such as phenolic acids, act as natural radical scavenging agents and metal chelators since they can neutralize free radicals via electron donation [28].

UPLC-ESI-QTOF-MS Metabolite Profiling of C. racemosa
After data processing, 49 potential molecular features were deconvoluted and annotated.Information on these molecular features (RT, m/z, formula molecular, relative abundances) is shown in Table 2.According to the identification guidelines proposed by Sumner et al. (2007) [29], 29 compounds were annotated based on their MS/MS spectra and compared with those spectra present in databases (level 2), 13 solely based on their molecular formula and MS 1 spectra (level 3), and 7 molecular features remained unknown (level 4).Different saccharides, amino acids, organic acids, fatty acids, phenolic compounds, and flavonoids were annotated in the analyzed extracts.LC/MS chromatograms of the methanolic extracts of C. racemosa leaves and flowers are depicted in Figure 1.
their antioxidant potential using three different models of antioxidant assays, viz., FRAP, DPPH, and ABTS assays.The results clearly indicated the antioxidant activities of both extracts (Table 1).However, the flower extract exhibited a significantly higher antioxidant capacity (IC50: 190 ± 8.0 µM TE/mg extract) for reducing Fe 3+ to Fe 2+ in an acidic medium in the FRAP assay compared to the leaf extract (IC50: 362 ± 24 µM TE/mg extract).Similarly, the flower extract showed a more potent radical scavenging capacity (IC50: 177 ± 2.0, 26 ± 2.0 µg/mL) than the leaf extract (IC50: 381 ± 2.0, 36 ± 2.0 µg/mL) using DPPH and ABTS methods, respectively.The unique diversity of flavonoid structures is what ultimately results in a marked difference in biological activities.In fact, flavonoids (kaempferol, luteolin, and apigenin), as well as non-flavonoid compounds such as phenolic acids, act as natural radical scavenging agents and metal chelators since they can neutralize free radicals via electron donation [28].

UPLC-ESI-QTOF-MS Metabolite Profiling of C. racemosa
After data processing, 49 potential molecular features were deconvoluted and annotated.Information on these molecular features (RT, m/z, formula molecular, relative abundances) is shown in Table 2.According to the identification guidelines proposed by Sumner et al. (2007) [29], 29 compounds were annotated based on their MS/MS spectra and compared with those spectra present in databases (level 2), 13 solely based on their molecular formula and MS 1 spectra (level 3), and 7 molecular features remained unknown (level 4).Different saccharides, amino acids, organic acids, fatty acids, phenolic compounds, and flavonoids were annotated in the analyzed extracts.LC/MS chromatograms of the methanolic extracts of C. racemosa leaves and flowers are depicted in Figure 1.   ) with a characteristic loss of CO 2 moiety [31].Protocatechuic and gallic acids were also previously reported in a characterization study of Colvillea based on HPLC-DAD [11].Notably, protocatechuic acid exerted antiproliferative effects on multiple cancer cell lines, viz., HL-60 leukemia, gastric adenocarcinoma, ovarian carcinoma A2780, and MCF-7 [39].

Flavonoids
Flavonoids are the most important polyphenolic secondary metabolites with diverse structures and functions.Based on MS/MS analysis, approximately 18 flavonoids belonging to different subclasses, viz., flavone-C-glycosides, flavones, flavonols, and isoflavones, have been identified.These compounds are presented mainly as glycosidic conjugates (Table 2).The linkage and the nature of sugars on the flavonoid skeleton occur either by attachment to the hydroxyl group, as in O-glycosides, or by direct linkage to the carbon atom in the A-ring, as in C-glycosides [40].

Flavone-C-Glycosides
In C. racemosa, luteolin and apigenin conjugates represented the unique landmarks of the plant.They were detected as mono or di-glycosides where the sugar substitution is typically present at C-6 and/or C-8.The key fragmentation patterns of apigenin glycosides showed the sequential loss of 120 and 90 amu for hexose and 90 and 60 amu for pentosyl residue, respectively.Peaks 42 min and 28.03 min) and were tentatively annotated as apigenin-8-C-glucoside (vitexin) and apigenin-6-Cglucoside (iso-vitexin), respectively [33].These results are in good agreement with those of Mohamed et al. (2018) [8], who isolated both compounds from the stems of C. racemosa.To the best of our knowledge, rhoifolin, luteolin 6-C-hexoside-8-C-arabinoside, apigenin-6-C-hexose-8-C-pentoside I, and apigenin-6-C-hexoside-8-C-pentoside II were identified for the first time in C. racemosa.Apparently, apigenin glycosides were previously reported to significantly inhibit cell proliferation and suppress lung cancer through downregulation of the IFN-γ signaling pathway [41,42].

Flavonols
Unlike C-flavonoids, most flavonoid-O-glycosides could be simply assigned by the neutral loss of sugar moieties, such as 162 amu for hexose, 146 amu for deoxyhexose, and 132 amu for pentose, resulting in the formation of their corresponding aglycones [40].Kaempferol-3-O-rutinoside (peak 26) was the most prominent flavonol identified at m/z 593.1511 (C 27 H 29 O 15 − ), which showed a characteristic loss of rutinoside unit to yield an intense peak at m/z 285 [37].Likewise, kaempferitrin (kaempferol di-rhamnoside) (peak 28) characteristically exhibited the molecular ion at m/z 577.1562 (C 27 H 29 O 14 − ) and showed its respective aglycone after one-step cleavage of two rhamnose molecules [36].Despite the previous identification of kaempferol aglycone in the stem and leaves of C. racemosa [8,11], the latter two glycosides were putatively identified for the first time in C. racemosa.Recently, kaempferol significantly inhibited the proliferation of lung cancer A549 cells through the downregulation of claudin-2 expression [43].Moreover, limocitrin (peak 29), a unique Citrus flavonol, was tentatively assigned based on its indicative fragment ions at m/z 345 and 330 [37].
Intriguingly, daidzein (peak 30) was the only isoflavone tentatively identified for the first time in Colvillea.It has been shown to play an important role in the regulation of cancer-related signaling pathways, especially in ovarian cancer [41].

Fatty Acids
Structurally, fatty acids (FAs) are long-chain hydrocarbons with reactive carboxylic acid groups.These compounds exhibited a potential broad-spectrum activity against cancer metabolism and proliferation [44].Oleic and linoleic acids (peaks 36  To assess the cytotoxic potential of the C. racemosa extracts, an SRB cytotoxicity assay was performed against the lung cancer cell line (A549).The results are shown in Figure 2.Both leaves and flower extracts inhibited the cell viability with IC 50 values of 17.0 and 17.2 µg/mL, respectively.Accordingly, the effect of leaf and flower methanolic extracts of C. racemosa on cell cycle distribution was assessed in the same cell line using flow cytometry, and the results are presented in Figure 3.The flower extract of C. racemosa displayed an increase in the cells in the S phase compared to the control (26.9 ± 1.0% to 27.9 ± 1.3%), suggesting S phase cell cycle arrest [45].Furthermore, a significant increase in the G2/M phase was also evoked by C. racemosa flower extract (15.2 ± 0.9% to 19.0 ± 1.6%).Interestingly, both leaf and flower extracts of C. racemosa showed a significant increase in the cells in the pre-G phase from 13.0 ± 0.2% to 15.3 ± 0.5% and 17.3 ± 0.3%, compared to the control group, respectively.a unique Citrus flavonol, was tentatively assigned based on its indicative fragment ions at m/z 345 and 330 [37].

Isoflavones
Intriguingly, daidzein (peak 30) was the only isoflavone tentatively identified for the first time in Colvillea.It has been shown to play an important role in the regulation of cancer-related signaling pathways, especially in ovarian cancer [41].

Fatty Acids
Structurally, fatty acids (FAs) are long-chain hydrocarbons with reactive carboxylic acid groups.These compounds exhibited a potential broad-spectrum activity against cancer metabolism and proliferation [44].Oleic and linoleic acids (peaks 36 and 38) were among the most abundant fatty acids detected at the end of the chromatographic run at m/z 281.2486 and 279.2329 with molecular formulas (C18H33O2 − ) and (C18H31O2 − ), respectively.

In Vitro Cytotoxicity against A549 Cell Line and Influence of C. racemosa Extracts on Cell Cycle Distribution
To assess the cytotoxic potential of the C. racemosa extracts, an SRB cytotoxicity assay was performed against the lung cancer cell line (A549).The results are shown in Figure 2.Both leaves and flower extracts inhibited the cell viability with IC50 values of 17.0 and 17.2 µg/mL, respectively.Accordingly, the effect of leaf and flower methanolic extracts of C. racemosa on cell cycle distribution was assessed in the same cell line using flow cytometry, and the results are presented in Figure 3.The flower extract of C. racemosa displayed an increase in the cells in the S phase compared to the control (26.9 ± 1.0% to 27.9 ± 1.3%), suggesting S phase cell cycle arrest [45].Furthermore, a significant increase in the G2/M phase was also evoked by C. racemosa flower extract (15.2 ± 0.9% to 19.0 ± 1.6%).Interestingly, both leaf and flower extracts of C. racemosa showed a significant increase in the cells in the pre-G phase from 13.0 ± 0.2% to 15.3 ± 0.5% and 17.3 ± 0.3%, compared to the control group, respectively.Previous studies highlighted the potential of flavonoid-C-glycosides as cytotoxic agents that affect cancer cell cycle distribution.A mechanistic study by Zhang et al., 2018) subsequently demonstrated that vitexin caused G2/M cell cycle arrest and, consequently, an accumulation of cell population was observed in this phase.This effect also caused G0/G1 to significantly decrease after treatment of the human glioblastoma cell line LN-18 with vitexin at the micromolar level [46].In this context, Yang and co-workers (2018) [47] reported that the cell cycle arrest at the G2/M phase was demonstrated by vicenin-2, also with a concurrent significant decrease in the G0/G1 phase.Importantly, the O-glycoside kaempferol rutinoside effectively induces intrinsic apoptosis in lung adenocarcinoma through mitochondrial dysfunction and calcium overload [48].Previous studies highlighted the potential of flavonoid-C-glycosides as cytotox agents that affect cancer cell cycle distribution.A mechanistic study by Zhang et al.,201 subsequently demonstrated that vitexin caused G2/M cell cycle arrest and, consequentl an accumulation of cell population was observed in this phase.This effect also cause G0/G1 to significantly decrease after treatment of the human glioblastoma cell line LNwith vitexin at the micromolar level [46].In this context, Yang and co-workers (2018) [4 reported that the cell cycle arrest at the G2/M phase was demonstrated by vicenin-2, al with a concurrent significant decrease in the G0/G1 phase.Importantly, the O-glycosid kaempferol rutinoside effectively induces intrinsic apoptosis in lung adenocarcinom through mitochondrial dysfunction and calcium overload [48].
Notably, isoorientin was reported to induce G2/M cell cycle arrest in the A549 c line.This effect, in addition to its apoptotic activity, was mediated via the signaling pat way of MAPK/STAT3/NF-κB, which supports its potential as a lead drug in lung canc management [49].
Nevertheless, orientin triggered cell cycle arrest at the G0/G1 phase in the HT-29 h man colorectal cancer cell line [50].These findings could further support the results metabolic profiling of C. racemosa extracts with vitexin, isoorientin, and vicenin-2 as t major detected flavonoid-C-glycosides, in addition to kaempferol rutinoside.Thus, t antiproliferative effect of Colvillea flower extract could be mediated by these major flav noid glycosides, mostly affecting the G2/M phase.Noteworthy, the increase in the dea cell population (pre-G phase) is indicative of cell death induced by the encompassed bi active compounds of C. racemosa.Notably, isoorientin was reported to induce G2/M cell cycle arrest in the A549 cell line.This effect, in addition to its apoptotic activity, was mediated via the signaling pathway of MAPK/STAT3/NF-κB, which supports its potential as a lead drug in lung cancer management [49].
Nevertheless, orientin triggered cell cycle arrest at the G0/G1 phase in the HT-29 human colorectal cancer cell line [50].These findings could further support the results of metabolic profiling of C. racemosa extracts with vitexin, isoorientin, and vicenin-2 as the major detected flavonoid-C-glycosides, in addition to kaempferol rutinoside.Thus, the antiproliferative effect of Colvillea flower extract could be mediated by these major flavonoid glycosides, mostly affecting the G2/M phase.Noteworthy, the increase in the dead cell population (pre-G phase) is indicative of cell death induced by the encompassed bioactive compounds of C. racemosa.

Docking Results
Molecular docking is an important computational technique to predict the best interaction between a ligand and its functional site [51,52].Phosphoinositide 3-kinases (PI3Ks) are a unique group of lipid kinases that represent the key regulators of many cellular functions, such as growth, metabolism, proliferation, differentiation, invasion, and immune signaling [23].Therefore, PI3K signaling is extensively studied as a promising target for combating cancer.Unambiguously, plant-based polyphenolic compounds have been reported to act as natural anticancer agents through a myriad of mechanisms, viz., inhibition of reactive oxygen species (ROS), activation of apoptosis, and suppression of protein kinases [14,53].Inspired by this concept, the most abundant flavonoids, viz., vitexin, kaempferol rutinoside, luteolin, and isoorientin, were analyzed on γ-PI3K through an in silico study, and the docking interactions are shown in Figure 4.As illustrated in Table 3, kaempferol rutinoside and vitexin showed the best binding scores among all investigated flavonoid compounds (−8.7140 and −7.5713 kcal/mol, respectively) compared to the docked co-crystallized inhibitor QYT (−6.6005 kcal/mol).They were stabilized into PI3K pocket through the formation of H-bonds with the amino acids Asp 964, Asp 950, Val 882, Ile 879, Ser 806, Thr 887, and Lys 890, rationalizing their potential activity as anticancer agents.Noteworthy, the docking results of kaempferol rutinoside are in line with those of Li et al. [48], who reported that kaempferol rutinoside, the unique flavone of Tetrastigma hemsleyanum successfully inhibited the cellular viability in A549 in a dose-dependent manner.
are a unique group of lipid kinases that represent the key regulators of many cellular functions, such as growth, metabolism, proliferation, differentiation, invasion, and immune signaling [23].Therefore, PI3K signaling is extensively studied as a promising target for combating cancer.Unambiguously, plant-based polyphenolic compounds have been reported to act as natural anticancer agents through a myriad of mechanisms, viz., inhibition of reactive oxygen species (ROS), activation of apoptosis, and suppression of protein kinases [14,53].Inspired by this concept, the most abundant flavonoids, viz., vitexin, kaempferol rutinoside, luteolin, and isoorientin, were analyzed on γ-PI3K through an in silico study, and the docking interactions are shown in Figure 4.As illustrated in Table 3, kaempferol rutinoside and vitexin showed the best binding scores among all investigated flavonoid compounds (−8.7140 and −7.5713 kcal/mol, respectively) compared to the docked co-crystallized inhibitor QYT (−6.6005 kcal/mol).They were stabilized into PI3K pocket through the formation of H-bonds with the amino acids Asp 964, Asp 950, Val 882, Ile 879, Ser 806, Thr 887, and Lys 890, rationalizing their potential activity as anticancer agents.Noteworthy, the docking results of kaempferol rutinoside are in line with those of Li et al. [48], who reported that kaempferol rutinoside, the unique flavone of Tetrastigma hemsleyanum successfully inhibited the cellular viability in A549 in a dose-dependent manner.Additionally, EGFR is another important target in cancer management for cancers associated with the overexpression of this receptor tyrosine kinase, like colorectal cancer.This prompted us to investigate the binding interaction potential of the most abundant flavonoids, viz., vitexin, kaempferol rutinoside, luteolin, and isoorientin with EGFR.Table 3 and Figure 5 display the docking findings of the examined compounds in comparison to the co-crystallized ligand AQ4 (S-score −8.5478 kcal/mol).Results showed that all four compounds demonstrated a good recognition of the active site amino acids with Sscores of −7.3955, −9.3078, −6.2791, and −8.0587 kcal/mol, respectively.Kaempferol rutinoside showed better binding than the co-crystallized ligand as reflected by its S-score forming a network of H-bonds with Ala 719, Asp 831, Arg 817, and Asn 818 active site amino acids.
Furthermore, CDK2 is also considered an important protein target in cancer [26].As such, we wanted to determine the potential effect of the selected flavonoids, viz., vitexin, kaempferol rutinoside, luteolin, and isoorientin on CDK2.In silico investigation, results reflected the ability of these flavonoids to recognize and interact with the key amino acids of the active site of CDK2 in a comparable manner to the co-crystallized inhibitor DTQ (Table 3 and Figure 6).While the co-crystallized ligand displayed an S-score for binding of −7.1365 kcal/mol, the investigated flavonoids had better or near S-scores of −7.5700, −8.5236, −6.3872, and −6.8739 kcal/mol, respectively.The best binder again was kaempferol rutinoside, forming two H-bonds with both Asp 145 and Val 18, in addition to a pi-H interaction with Ala 144.
Details of the 3D interactions of the most active compound in the docking study by virtue of its high binding S-scores, kaempferol rutinoside, are presented in Figure 7.With all three biological targets, PI3K, EGFR, and CDK2, kaempferol rutinoside was able to establish a network of H-bonds and pi-H bonding interactions with the active site amino acids to stabilize its complex with these biotargets and hence elicit its inhibitory activity paving the way to the anticancer effect of C. racemosa it participates to.Furthermore, CDK2 is also considered an important protein target in cancer [26].As such, we wanted to determine the potential effect of the selected flavonoids, viz., vitexin, kaempferol rutinoside, luteolin, and isoorientin on CDK2.In silico investigation, results reflected the ability of these flavonoids to recognize and interact with the key amino acids of the active site of CDK2 in a comparable manner to the co-crystallized inhibitor DTQ Ultimately, our docking study provides scientific evidence for the promising anticancer activity of C. racemosa.
On the other hand, aside from our in vitro and in silico findings on the cytotoxic potential of C. racemosa extracts, extrapolation on in vivo testing will be our future perspective.Also, detailed risk assessment and systemic toxicity profile should be investigated to guard against any cytotoxic effects on normal tissues.Additionally, after sufficient in vivo exploration, the synergistic effect of C. racemosa extracts with drugs used for NSCLC will be highly valuable for decreasing the side effects of these synthetic drugs.
(Table 3 and Figure 6).While the co-crystallized ligand displayed an S-score for binding of −7.1365 kcal/mol, the investigated flavonoids had better or near S-scores of −7.5700, −8.5236, −6.3872, and −6.8739 kcal/mol, respectively.The best binder again was kaempferol rutinoside, forming two H-bonds with both Asp 145 and Val 18, in addition to a pi-H interaction with Ala 144.Details of the 3D interactions of the most active compound in the docking study by virtue of its high binding S-scores, kaempferol rutinoside, are presented in Figure 7.With all three biological targets, PI3K, EGFR, and CDK2, kaempferol rutinoside was able to establish a network of H-bonds and pi-H bonding interactions with the active site amino acids to stabilize its complex with these biotargets and hence elicit its inhibitory activity paving the way to the anticancer effect of C. racemosa it participates to.Ultimately, our docking study provides scientific evidence for the promising anticancer activity of C. racemosa.
On the other hand, aside from our in vitro and in silico findings on the cytotoxic potential of C. racemosa extracts, extrapolation on in vivo testing will be our future perspective.Also, detailed risk assessment and systemic toxicity profile should be investigated to guard against any cytotoxic effects on normal tissues.Additionally, after sufficient in vivo exploration, the synergistic effect of C. racemosa extracts with drugs used for NSCLC will be highly valuable for decreasing the side effects of these synthetic drugs.

UPLC-ESI-QTOF-MS Metabolite Profiling of Leaves and Flowers of C. racemosa
Flower and leaf extracts of C. racemosa were analyzed using an ACQUITY UPLC H-Class System (Waters, Milford, MA, USA) coupled to a QTOF-MS (Synapt G2, Waters Corp., Milford, MA, USA).A reversed-phase C18 column (Agilent Zorbax Eclipse Plus, 2.1 × 150 mm, 3.5 µm) was used for the chromatographic separation.The mobile phases were acidified water with acetic acid at 0.5% (phase A) and methanol (phase B).The following mobile phase gradient was established for optimal chromatographic separation: 0 min (100% A), 5 min (75% A), 20 min (40% A), 38 min (0% A), and 46-56 min (100%A).The flow rate, column temperature, and injection volume were set at 400 µL/min, 22 • C, and 10 µL, respectively.Mass detection was performed over a range of mass to charge ratio (m/z) from 50 to 1200, using negative electrospray ionization mode (ESI-).MS acquisition was carried out using two scan functions by fast switching, in which one of them was operated with a low collision energy in the gas cell (4 eV) and the other with a high collision energy using a ramp from 20 to 60 eV (MSE mode).Leu-Enkephalin was continuously injected for mass calibration.Other MS parameters were as follows: desolvation temperature 500 • C; source temperature 100 • C; desolvation gas flow 700 L/h; cone gas flow 50 L/H; scan; capillary voltage 2.2 kV; cone voltage 30 V; duration 0.1 s; and resolution 20,000 FWHM.

MS Data Processing
The raw data files were first converted to .mzMLformat using MSConver GUI software (Proteowizard; https://proteowizard.sourceforge.io/(accessed on 15 January 2024)) [54].The MS data processing steps were carried out using MZmine 2.53 [55].The following parameters were set for the different stages.(  Stock solutions of gallic acid and rutin in methanol (1 mg/mL) were prepared, and seven serial dilutions were acquired in the concentrations of (50, 100, 200, 400, 600, 800, 1000) µg/mL, and standard calibration curves were constructed accordingly.The leaf and flower extracts of C. racemosa were prepared at concentrations of 4 mg/mL in methanol.Each of the standards and two plant extracts were pipetted into the plate wells in six replicates.Measurements were performed at 630 and 420 nm for phenolics and flavonoids, respectively, using a FluoStar Omega microplate reader (BMG LABTECH, Ortenberg, Germany) [57,58].

FRAP Assay
The FRAP assay was established according to the standard Fe 3+ reducing power assay of Benzie and Strain [59] with some modifications to be suitable for microplates.Briefly, solutions of leaves and flowers of C. racemosa were prepared in a concentration of 1 mg/mL in methanol.Similarly, Trolox stock solutions (10 mg/mL) in methanol with serial dilutions were prepared at concentrations of 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 µg/mL.The 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) reagent was freshly prepared (300 mM acetate buffer (pH = 3.6), 10 mM TBTZ in 40 mM HCl, and 20 mM FeCl 3 , in a ratio of 10:1:1 v/v/v, respectively.TPTZ (190 µL) was mixed with 10 µL of the investigated Colvillea extracts in a 96-well plate (n = 6), and the reaction mixture was incubated at room temperature (RT) for 30 min in darkness.Finally, the absorbance of triplicate samples was measured at 593 nm using a FluoStar Omega microplate reader (BMG LABTECH, Ortenberg, Germany).The ferric-reducing ability is presented as µM TE/ mg dried extract.

DPPH Assay
A DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay was performed to evaluate the free radical scavenging potential of the Colvillea extracts as previously described by Boly et al. (2016) [60].Briefly, solutions of the leaves and flowers of C. racemosa (1 mg/mL) were serially diluted to provide five concentrations (400, 300, 200, 100, and 50 µg/mL).DPPH reagent 100 µL (0.1%) was added to 100 µL of the extract in a 96-well plate (n = 6).The reaction mixture was incubated at room temperature for 30 min under darkness.After incubation, the absorbance was measured at 540 nm.Each sample was measured in triplicate.The results were acquired using a FluoStar Omega microplate reader.The data were analyzed using Microsoft Excel 2016 ® , and the IC 50 value was calculated using GraphPad Prism 8 ® [61].

ABTS Assay
The ABTS assay was carried out as previously described by Arnao et al. (2001) [62], with some modifications.Briefly, several concentrations of leaves and flowers of C. racemosa were prepared (100, 200, 400, 600, and 800 µg/mL) from a stock solution of 1mg/mL in methanol.One milliliter of ABTS solution (192 mg/50 mL distilled water) was added to 17 µL of 140 mM potassium persulfate and left in the dark for 24 h.Then, 1 mL of the reaction mixture was diluted to 50 mL, and 190 µL of the freshly prepared ABTS reagent was mixed with 10 µL of C. racemosa extract in a 96-well plate (n = 6).The reaction mixture was incubated at room temperature for 120 min in the dark.The absorbance was measured at 734 nm.The sulforhodamine B (SRB) assay was a well-established method for screening the cytotoxicity of plant extracts.In brief, different concentrations (0.01, 0.1, 1, 10, and 100 µg/mL) of both leaf and flower extracts from C. racemosa were used.Lung cancer cell lines (A-549) were obtained from Nawah Scientific Inc. (Cairo, Egypt).The cells were maintained in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO 2 atmosphere at 37 • C. Cell viability was evaluated by SRB assay [63,64].Aliquots of 100 µL cell suspension (5 × 10 3 cells) were incubated in 96-well plates for 24 h.Then, the suspension was treated with 100 µL media containing the extracts at various concentrations ranging from (0.01, 0.1, 1, 10, and 100 µg/mL) for 72 h.Fixation of the cells was performed by replacing the media with 150 µL of 10% TCA and incubated at 4 • C for 1 h.Finally, the TCA solution was removed, and the cells were washed with distilled water, followed by the addition of 70 µL of SRB solution (0.4% w/v) and incubated at room temperature for 10 min in the dark.The plates were washed with 1% acetic acid (3 times) and left to dry overnight.Then, 150 µL of 10 mM Tris was added to solublize protein-bound SRB stain, and the absorbance was measured at 540 nm by a BMG LABTECH ® -FLUOstar Omega microplate reader (Ortenberg, Germany).

Analysis of Cell Cycle Distribution
The cells were treated with leaf and flower extracts of C. racemosa for 48 h.Cells (105 cells) were collected by trypsinization and then washed twice with ice-cold PBS (pH 7.4).The cells were re-suspended in 60% ice-cold ethanol (2 mL) and incubated at 4 • C for 1 h to allow fixation.Fixed cells were washed with PBS (pH 7.4) twice and suspended in 1 mL of PBS containing 50 µg/mL of RNAase A and 10 µg/mL of propidium iodide.After 20 min, the DNA contents of the cells were analyzed via flow cytometry analysis using an FL2 (λex/em 535/617 nm) signal detector (ACEA Novocyte™ flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA).For each sample, 12,000 events were recorded.Cell cycle distribution was calculated using the software of ACEA NovoExpress™ version 1.1.0(ACEA Biosciences Inc., San Diego, CA, USA) [45,65].

Statistical Analysis
The results of antioxidant, in-vitro cytotoxicity, and cell cycle analyses are presented as the mean ± SEM using GraphPad Prism 8 software.One-way ANOVA followed by Tukey's post hoc test was used to test the significance; p < 0.05 was taken as the cut-off value for significance.

Conclusions
The current study describes the detailed metabolic profiling of flower and leaf extracts of Colvillea racemosa, which resulted in the putative annotation of 42 metabolites, including saccharides, phenolic acids, flavonoids, amino acids, and fatty acids.The antioxidant activity and cytotoxicity against non-small cell lung cancer cell lines were demonstrated in this study for both extracts, highlighting their cytotoxic potential.The C-glycoside flavonoids characterized in both extracts stand out due to their high abundance and the properties previously reported for this class of compounds.The docking results of four major flavonoids showed that kaempferol-3-O-rutinoside and vitexin had the highest binding scores on γ-PI3K and CDK2, followed by isoorientin.Meanwhile, kaempferol-3-O-rutinoside and isoorientin exhibited the best binding scores on EGFR.Thus, our docking results rationalize the potential in vitro activity of C. racemosa extracts as cytotoxic agents against NSCLC.Despite these promising results, more studies are needed to isolate these potentially active ingredients and to reveal the corresponding extensive potential mechanism of action, which allows its use in possible future therapeutic applications.

Figure 1 .
Figure 1.LC/MS base peak chromatograms of methanolic extracts of C. racemosa leaves (A) and flowers (B).

Figure 1 .
Figure 1.LC/MS base peak chromatograms of methanolic extracts of C. racemosa leaves (A) and flowers (B).

Figure 2 .
Figure 2. In vitro cytotoxic potential of leaf (A) and flower (B) extracts of Colvillea racemosa against A549 lung cancer cell line (IC50 values are shown in the graphs).Figure 2. In vitro cytotoxic potential of leaf (A) and flower (B) extracts of Colvillea racemosa against A549 lung cancer cell line (IC 50 values are shown in the graphs).

Figure 2 .
Figure 2. In vitro cytotoxic potential of leaf (A) and flower (B) extracts of Colvillea racemosa against A549 lung cancer cell line (IC50 values are shown in the graphs).Figure 2. In vitro cytotoxic potential of leaf (A) and flower (B) extracts of Colvillea racemosa against A549 lung cancer cell line (IC 50 values are shown in the graphs).

Figure 3 .
Figure 3. Diagrammatic representation of cell cycle analysis using flow cytometry following 24 incubation of leaf and flower extracts of Colvillea racemosa at their IC50 with A549 lung cancer c line.

Figure 3 .
Figure 3. Diagrammatic representation of cell cycle analysis using flow cytometry following 24 h incubation of leaf and flower extracts of Colvillea racemosa at their IC 50 with A549 lung cancer cell line.

3 .
Materials and Methods 3.1.Plant Material Leaves and flowers of C. racemosa Bojer ex Hook were collected in July 2019 from Mostafa EL-Abd Gardens, Cairo-Alexandria Desert Road, Egypt (30.3552330N, 30.5166490W).The plant material was kindly identified by Mrs. Therese Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture and Orman Botanical Garden, Giza, Egypt.Voucher specimen (No. 10.7.2019) was kept in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University.Leaves and flowers were air-dried in the shade, powdered, and stored in airtight containers until subsequent analysis.A stock solution of each C. racemosa extract (5 mg/mL) was prepared for UPLC-ESI-QTOF-MS analysis.

Table 1 .
Quantitative determination of total phenolic and flavonoid contents and in-vitro antioxidant assays of leaves and flowers of C. racemosa extracts.

Table 2 .
Metabolite profiling of the methanolic extracts of leaves and flowers of C. racemosa as analyzed by UPLC-ESI-QTOF-MS.

Table 2 .
[39].Phenolic acids are among the simplest secondary metabolites formed of one phenolic ring with multiple hydroxy or methoxy groups and display high potential for use as anticancer agents with diverse targets[39].Benzoic acid derivatives, either as aglycones or glycosidic conjugates, were the most abundant phenolic acids identified in the flowers and leaves of C. racemosa.Protocatechuic acid and dihydroxybenzoic acid pentoside displayed [M − H] − at m/z 153.0193 (C 7 H 5 O 4 − ) and 285.0615 (C 12 H 13 O 8 − and 38) were among the most abundant fatty acids detected at the end of the chromatographic run at m/z 281.2486 and 279.2329 with molecular formulas (C 18 H 33 O 2 − ) and (C 18 H 31 O 2 − ), respectively.2.4.In Vitro Cytotoxicity against A549 Cell Line and Influence of C. racemosa Extracts on Cell Cycle Distribution

Table 3 .
Molecular docking scores of important metabolites of Colvillea racemosa on different protein targets involved in lung cancer.