Introduction

All over the world, cancer ranks as a primary cause of mortality and a significant roadblock to raising life expectancy [1, 2]. According to World Health Organization (WHO) estimations for 2022, globally cancer represented the cause of death for 16% before the age of 70 [3]. Hepatocellular carcinoma is the predominant primary cancer in most countries and the fourth most prevalent cancer across the globe [4, 5] besides being the third most lethal cancer-associated mortality in the world [6].

Additionally, breast cancer represents the first-leading cause of death for women, almost 2.3 million women received a breast cancer diagnosis in the world in 2020, and 685,000 of them passed away. Somewhere in the globe, a woman receives a breast cancer diagnosis every 14 s [6, 7]. The main regimen of treatment of various forms of cancer is to stop unregulated cell growth which can be achieved by using cytotoxic drug medications. The effect of these drugs can be estimated by using cell-based in vitro assays to measure the degree of tissue-level cell damage [8].

However, the use of conventional chemotherapeutic agents has been associated with a wide range of side effects and toxicities; therefore, new approaches for the prevention and cure of cancer represent a great challenge for researchers [9]. One of the most crucial methods for treating particular types of cancer is the discovery of natural anti-cancer medications, which requires constant monitoring of various sources such as marine animals, terrestrial plants, and seaweeds [10].

There are more than 60 species of halophyte plants in the genus Tamarix  belonging to the Tamaricaceae family, which are cultivated in almost every region of the world under the common names “Tamarisk” and “salt cedar” [11, 12]. It has a variety of therapeutic uses in conventional medicine [11]. Due to the plant’s astringent and cleaning properties on internal organs, which were attributed to its bitter taste, it was known to have a chilly and dry nature [11]. Certain Tamarix species are recommended as mild laxatives, anti-tussive, antipyretics, and tonics for the liver and spleen [11, 13]. Some species are used to treat leucorrhea and uterine bleeding because they have anti-inflammatory and wound-healing characteristics [14]. It can be applied topically to treat skin conditions like eczema and anal fissure [13]. Biological studies have demonstrated that some Tamarix species can be used as anti-Alzheimer [15], anti-diabetic [16], anti-hyperlipidemic [17], anti-inflammatory [18, 19], antimicrobial [20, 21], antinociceptive [22], antioxidant [23], anti-coagulation [24], anti-rheumatoid [25], cytotoxicity [26], hepatoprotective [27] and wound healing [28] activities. Tamarix is represented in Egypt with two indigenous species which are T. aphylla (L.) H. Karst and T. nilotica (Ehrenb.) Bunge. T. nilotica is a rich source of polyphenolics including hydrolyzable tannins, sulfated and non-sulfated flavonoids, and phenylpropanoids [29, 30]. T. nilotica extracts have demonstrated antioxidant, antiangiogenic, cytotoxic, hepatoprotective, antifibrotic, antidiabetic, and antimicrobial activities in relation to their phenolic contents [29,30,31]. Although both species are indigenous in Egypt, many studies targeted T. aphylla which was mentioned for comparison to T. nilotica [16, 20, 22, 28, 32,33,34,35]. Besides, T. nilotica was the one easily available for us to carry on with this study.

In the previous published studies, T. nilotica received much attention in studying its cytotoxic activity. Various studies reported the effect of leaves, methanolic flower extracts on different cell lines including lung (A-549), liver (Huh-7), colon (HCT-116), and breast (MCF-7) cancer cell lines [36,37,38]. T. nilotica flower extract reported to exhibit hepatoprotective and antioxidant activities [38]. However, there are no studies concerning the cytotoxic activities of the n-butanol fraction of T. nilotica flower.

The present work aimed to investigate the possible cytotoxic activity of the n-butanol extract of T. nilotica flowers against liver (Huh-7) and breast (MCF-7) cell carcinoma while performing an in-depth phytochemical analysis of the same extract n-butanol extract using LC-MS/MS analysis to relate the activity to the extract’s metabolites.

Methods

Statement

All experiments and methods including the collection of the plant were performed following the relevant national, and international guidelines and legislation of the Faculty of Pharmacy, University of Sadat City, Sadat City, Egypt.

Extraction and Isolation

The air-dried flowers of T. nilotica (Ehrenb.) Bunge (1 kg) was exhaustively extracted with 80% methanol; excess solvent was removed using a rotary evaporator. The crude aqueous methanolic extract was further fractionated using solvents of different polarity viz., n-hexane, dichloromethane, n-butanol, and water. The fractions were dried under vacuum to give their corresponding weights of 30 gm, 25 gm 15 gm, and 45 gm, respectively. All fractions were stored at -20 °C till further analysis [39].

LC–LTQ–MS–MS analysis

The n- butanol extract was analyzed and processed using LC–MS–MS. A Shimadzu LC-10 HPLC with a Grace Vydac Everest Narrowbore C-18 column (100 mm × 2.1 mm i.d., 5 μm, 300 Å). An LC–MS, connected to an LTQ Linear Ion Trap MS (Thermo Finnigan, San Jose, CA) was utilized with a mass range of 100–2000 m/z. A 2 µL sample was injected using an autosampler. A 35 min method was used as follows: 5 min isocratic run using 5% acetonitrile (Acn) and 0.05% formic acid (FA), then a gradient was run for 25 min until 95% AcN 0.05% FA. Finally, there was 5 min of conditioning the column with 5% AcN and 0.05% FA. The data were processed and analyzed using foundation 3.1_Xcalibur_3.1.6610 as well as MZmine3. Furthermore, the raw data files were converted to mzXML format using MSConvert from the ProteoWizard suite [40]. The molecular network was created using the Global Natural Products Social Molecular Networking (GNPS) online workflow. The spectra in the network were then searched against the GNPS spectral libraries and published data [41, 42].

Using the GNPS dataset, the raw MS file was analyzed. By analyzing the similarity between the fragmentation pattern from the raw mass spectrum and the GNPS library, GNPS assists in the identification and discovery of metabolites. Other installed programs, including MSConvert (https://proteowizard.sourceforge.io/), File Zilla (https://filezilla-project.org/), and Cytoscape version 3.5.1(https://cytoscape.org/), were used to operate with GNPS at the following link (https://gnps.ucsd.edu/) [43, 44].

1H-NMR analysis

1H-NMR spectra were recorded at 298 K on a Bruker 600 MHz (TCI CRPHe TR-1H and 19F/13C/15N 5 mm-EZ CryoProbe) spectrometer. Chemical shifts were referenced to the solvent peak for CH3OD at δH 3.3100 ppm [44, 45].

Cytotoxic evaluation of the n-butanol fraction of T. nilotica flowers

Cell cultures

Breast adenocarcinoma cell lines (MCF-7) and hepatocyte-derived cellular carcinoma cell lines, human liver (Huh-7) was obtained from Nawah Scientific Inc., (Mokatam, Cairo, Egypt). 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 humidified, 5% (v/v) CO2 atmosphere at 37 °C [46].

Cell cytotoxicity

Cell viability was assessed by sulforhodamine B (SRB) assay on two cancer cell lines [47, 48], the human liver cancer cell line (Huh-7) and the breast cancer cell line (MCF-7). Aliquots of 100 µL cell suspension (5 × 103^ cells) were in 96-well plates and incubated in complete media for 24 h. Cells were treated with another aliquot of 100 µL media containing the n-butanol T. nilotica flower extract at two different concentrations (10 and 100 µg/ml). After 72 h, cells were fixed by replacing media with 150 µL of 10% TCA and incubated at 4 °C for 1 h. 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 (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) [49]. The cell morphological analysis was carried out according to M. Roy et al. 2017 [50].

Statistical analysis

Statistical analysis of the data was performed using one-way ANOVA, followed by Tukey’s multiple range tests for post hoc comparisons (GraphPad Prism, version 8.4.2). All the data are presented as the means of 3 determinations ± SE [51].

Results

Metabolic profiling of the n-butanol fraction of T. nilotica flowers using LC–LTQ–MS–MS analysis in positive mode

Based on the exact mass, the observed spectra fragmentation patterns, and literature data, the structural characterizations of chemical composition in the n-butanol fraction of the T. nilotica flowers were accomplished. Using MS/MS fragmentation pattern, 39 compounds from various classes of secondary metabolites were identified. The detected compounds’ structures were presented in (Fig. 1). Molecular ion, retention time, and MS/MS data of identified compounds were provided in (Table 1).

Table 1 Metabolites tentatively identified from the n-butanol fraction of T. nilotica flowers using LC–LTQ–MS–MS analysis in positive mode
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Fig. 1
figure 1

Chemical structures of the tentatively identified compounds in the n-butanol fraction of T. nilotica flowers numbered according to compounds listed in Table 1  

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LC–LTQ–MS–MS analysis of the n-butanol fraction of T. nilotica flowers using GNPS-Aided annotation

Metabolite profiling of the n-butanol fraction of T. nilotica flowers via GNPS based on tandem mass spectrometry data as well as a dictionary of natural products yielded the annotation of 35 metabolites (N1—N35); mainly flavonoids, phenolics, and fatty acids; respectively (Figs. 1 and 2; Table 2). Flavonoids were annotated by observing the common fragments of retro dials-alder reaction indicated at m/z 153, 152, 135 depending on structure as in N11, 15, 16, 17, 18, etc. Additionally, common fragments such as [M-18 Da] denoting loss of H2O molecule, [M-28 Da] denoting the loss of CO, [M + H-42]+ corresponding to C2H2O loss, besides [M + H-46]+, as in quercetin, kaempferol, and myricetin derivatives. A common fragment in O-methylated flavonoids is [M + H-15]+ formed by loss of methyl radical, as shown in N10 (Kaempferide-O-hexoside), N21 (Kaempferide-O-hexoside derivative), N28 (kaempferide), N20 (tamarixetin), N32 (kaempferol 4’,7-dimethyl ether), N30 (quercetin- dimethyl ether) and N18 (herbacetin-trimethyl ether). Flavanones were annotated in the form of dihydro derivatives of flavonols as presented in N26 (m/z 305) tentatively identified as dihydro-quercetin, N31 (m/z 321) identified as dihydromyricetin. Phenolic acids i.e., N5, N12, N13, and N24 were previously reported in Tamarix species. GNPS databases also aided in identifying N7, N9, N14, N25, and N34, besides kaempferol derivatives as well (Fig. 3).

Table 2 Metabolites identified from the n-butanol fraction of T. nilotica flowers based on NMR and GNP analysis. No. = numbers of identified metabolites, Rt= retention time in mins, MF = molecular formula, ID = name of identified compounds, Ref. = references of identified compounds
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Fig. 2
figure 2

LTQ-LC-MS-MS chromatogram of the n- butanol fraction of T. nilotica flowers

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Fig. 3
figure 3

Molecular network (showing clusters of metabolites of interest) based on tandem mass spectrometry data in the positive ionization mode of the n-butanol fraction of T. nilotica flowers. Twenty metabolites have been identified as labeled in Fig. 3, green color indicating the number of compounds in Table 2, light blue nodes are compounds identified using GNPS databases, while the identified compounds using fragmentation matching have the pink color

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Nuclear magnetic resonance (NMR) analysis

To provide a broader scope of the n-butanol fraction T. nilotica flowers metabolome, 1H-NMR was used to provide insights into both secondary and primary metabolites that were not detected by LTQ-LC-MS-MS. 1H-NMR can also be used for structural elucidation and determining major metabolites. Sugars, flavonoids, phenolics, and coumarins were among the major metabolites classes detected in the n-butanol fraction of T. nilotica flowers using 1H-NMR as detailed in (Table 3).

Table 3 The identified metabolites of the n-butanol fraction of T. nilotica flowers exhibited at 1H-NMR
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Fatty acids were discriminated against by the presence of terminal (CH3 ) at δH 0.9 ppm, long chain methylene groups at δH 1.2 ppm, and olefinic (CH) showed at δH 5.3 ppm, as shown in (Fig. 4, M1).

Sugars, the second intense metabolites, were recognized by the presence of anomeric proton annotated as, α, β glucose, and sucrose, which exhibited anomeric protons at δH 5.18 (d, J = 3.8 Hz) for (Fig. 4, M2), δH 4.58 (d, J = 7.8 Hz) (Fig. 4, M3), and δH 5.40 (d, J = 3.8 Hz), δH 4.17 (d, J = 8.5 Hz) (Fig. 4, M4), respectively. Moreover, CHs attached to hydroxyl groups exhibited overlapped peaks at a range of δH 3.2—4.02 ppm as shown in (Fig. 4, M2-M4) [91]. A sharp singlet peak at δH 2.56 (s) indicated the presence of a common organic acid elucidated as succinic acid (Fig. 4, M5) [91]. Finally, flavonoids and coumarins were found in a region of aromaticity, which was recognized by the presence of δH 6.35, 7.60 (d, J = 15.8 Hz) corresponding to α, β unsaturated ketone in coumarins. Concerning flavonoids overlapped peaks at the region of δH 6.0—8.33 ppm, which was elucidated with the help of LTQ-LC-MS-MS data (Fig. 5).

Fig. 4
figure 4

1H-NMR spectrum exhibiting the identified metabolites in the n-butanol fraction of T. nilotica flowers; primary metabolites i.e., fatty acids and sugars (M1-M4) as well as organic acid (M5) at the aliphatic region δH 0.5—5.5 ppm as mentioned in Table 3

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Fig. 5
figure 5

1H-NMR spectrum exhibiting the identified metabolites in the n-butanol fraction of T. nilotica flowers; in aromatic region δH 5.5—8.2 ppm prescribing coumarins and flavonoids

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Cytotoxic evaluation of the n-butanol fraction of T. nilotica flowers

The cytotoxic effect of the n-butanol fraction T. nilotica flowers was investigated as a cytotoxicity SRB quick screening against MCF-7 and Huh-7 cells. The n-butanol fraction inhibited cancer cells in a dose-dependent manner since the activity increased with increasing the dose. For instance, at a concentration of 100 µg/ml, the viability percentage was 54.27% compared to 100% with 10 µg/mL on MCF-7 with an IC50 ˃100 µg/mL. However, the best effect was observed with Huh-7 where the percentage viability decreased from 51.89% at 10 µg/mL to 7.22% at 100 µg/mL with an IC50 = 37 µg/mL (Table 4).

Cell viability was assessed at five different concentrations (0.01, 0.1, 1, 10, and 100 µg/mL) using the SRB assay revealed that T. nilotica flowers n-butanol fraction possesses a dose-dependent cytotoxic effect with an IC50 of 37 µg/mL with Huh-7 cell lines while it showed IC50 > 100 µg/mL with MCF-7 cell lines (Fig. 6).

Table 4 Cytotoxicity SRB quick screening results of the n- butanol fraction of T. nilotica flowers
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Fig. 6
figure 6

In-vitro SRB cytotoxicity assay of the n-butanol fraction of T. nilotica flowers against A: Huh-7 and B MCF-7 cell lines in increasing concentrations (0.01–100 µg/mL). Data points are expressed as mean ± SD (n = 3)

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Fig. 7
figure 7

Optical microscope-stained images of quick screening SRB cytotoxicity assay of the n- butanol fraction of T. nilotica flowers against MCF-7; A: negative control, B: 10 µg/mL, C: 100 µg/mL, and Huh-7; D: negative control, E: 10 µg/mL, F: 100 µg/mL

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Discussion

One of the leading causes of death on the globe is cancer. Given their significant toxicity to cancer cells, natural products, and their secondary metabolites are highly significant for research into potential anticancer treatments. Previous research found that several Tamarix species have displayed varying cytotoxic activities. Breast adenocarcinoma cells (MCF-7) were suppressed by the methanolic extract of T. aphylla in a concentration-dependent manner [33]. Different extracts of T. senegalensis demonstrated anti-cancer effects in human liver (Huh-7) and lung (A-549) carcinoma cells [31]. T. gallica shoots, flowers, and leaves methanolic extracts were able to inhibit the proliferation of colon cancer (Caco-2) cells at concentrations of 50 and 100 g/mL [82]. Furthermore, T. articulata methanolic extract demonstrated promising antiproliferative activity against hepatocellular carcinoma [92], as well as against prostate cancer (LnCaP) cells’ motility and invasiveness in a dose-dependent manner [93]. In this study, the n-butanol fraction of T. nilotica flowers showed cytotoxic activity against MCF-7 and Huh-7 cells (Fig. 6) in a dose-dependent manner with a more promising effect against liver cancer cell Huh-7 (IC50 = 37 µg/mL). The optical microscope-stained images were recorded as shown in Fig. 7 comparing the cytotoxic effect of n-butanol fraction of T. nilotica flowers at a concentration of 10 and 100 µg/mL with comparison to (-ve control). Images clearly show the cytotoxic effect of the extract against MCF-7 and Huh-7 cell lines (Fig. 7C, E & F) where no morphological changes were observed on MCF-7 at conc. 10 µg/mL (Fig. 7B) as well as the negative control of both cell lines (Fig. 7A & D) while more potent effect was observed against Huh-7 (Fig. 7E & F). This confirms that the n-butanol fraction of T. nilotica flowers possess cytotoxic effects which are clearer and more potent on Huh-7 cells over MCF-7 cells.

T.nilotica has been previously reported for promising cytotoxic activity against human colon (HCT-116) and breast (MCF-7) cancer cells [94], whereas ethyl acetate was active against lung cancer cell line with increased expression levels of p-53 and Bax whereas that of Bcl-2 was decreased [36, 37], while flowers were effective and selective against liver cell carcinoma (Huh-7) [38].

The chemical investigation of various Tamarix species was reported. Gallic acid, flavones, and flavonols were among the polyphenols found in this study that were recognized as compounds that had previously been found in other species of Tamarix [34, 95]. For example, a study on the alcohol-soluble fraction of an aqueous extract of T. nilotica aerial parts collected from Egypt and Saudi Arabia was discussed by Sekkien A. et al. 2018 [30]. The study reported that the major compounds in the Egyptian species extract were (iso)ferulic acid-3-sulphate, methyl ferulate sulfate, and coniferyl alcohol sulfate derivative. Moreover, this species exhibited the presence of kaempferide, gallic acid, nilocitin, kaempferol dimethyl ether sulfate, tamarixetin, kaempferol, quercetin, methyl gallate methyl ether, kaempferol 3-O-β-glucuronide and 4ʹ-O-methyl quercetin 3-O-β-hexoside which was following the identified compounds in our study [30]. Also, the tannin-identified compounds in our study as hirtellin B, gemin D, nilotinin D1, and tamarixinin A were following those reported in T. nilotica, T. pakistanica, T. tetrandra, and T. senegalensis by [56, 64, 68, 96]. These several identified polyphenolic compounds in this genus explain its widespread biological activity as stated in [11].

The phytochemical analysis of the n-butanol extract of T. nilotica flowers using LC-MS/MS analysis reveals the identification of various phenolic compounds such as gallic acid, caffeic acid, ferulic acid, luteolin, kaempferol, quercetin, kaempferol-3-O-glucuronide, tamarixetin, besides various galloyl and gallate moieties. Fragments at m/z [M-H-152]− and [M-H-170] – denoted the losses of galloyl and gallate moieties respectively, eliminated by gallotannins or galloylated esters [60]. Tannins were previously isolated and identified in T. nilotica and have shown potent cytotoxic effects with high tumor specificity [68]. The promising cytotoxic effect against liver carcinoma can be well correlated with the tentatively identified phenolic compounds where caffeic and gallic acid was reported to reduce the growth of MCF-7 breast cancer cells and altered the expression of apoptotic genes [97], ferulic acid also promotes apoptosis in cancer cell lines MCF-7 and HepG-2 and activated the caspase-8 and − 9 pathways, has cytotoxic action and [98]. while nilocitin showed a G2/M and S cell cycle arrest as a consequence of the G1 phase [99], furthermore, the flavonoid hispidulin (4’,5,7-trihydroxy-6-methoxyflavone) causes ERS-mediated apoptosis in hepatocellular carcinoma cells by stimulating the AMPK/mTOR pathway, [100]. HepG-2 cells were more vulnerable to hispidulin-mediated cell death than immortalized L929 fibroblasts, indicating that this substance has a distinct level of toxicity in tumor-related cell lines than normal cell lines [101]. When kaempferol was administered to the human breast cancer cell line MCF-7, it suppressed the expression of PLK-1, a protein-like kinase that has been shown to control mitotic development and to be elevated in several human cancers. Kaempferol’s anticancer activity is mediated via inhibition of the EGFR-related Src, ERK1/2, and AKT pathways, and it may be a powerful inhibitor of pancreatic cancer cells [102]. Luteolin is a very significant flavonoid that is present in many foods. It has several health benefits, including its ability to prevent cancer, induce cell cycle arrest and apoptosis in some human cancer cells, and enhance the antitumor effects of 5-FU on Bel7402 and HepG-2 cells. These effects may be connected to apoptosis and the control of 5-FU metabolism [103,104,105]. The dietary flavonoid quercetin, which is found in berries, demonstrated high cytotoxicity it prevented HepG-2 cancer cells from proliferating and surviving while inducing apoptosis by increasing the expression of p53 and BAX [106, 107].

Our findings imply that the T. nilotica flower’s n-butanol fraction has the potential to be a promising cytotoxic candidate against Huh-7 cancer cells.

Conclusion

This study documents a detailed metabolites profiling for the unexplored n-butanol fraction of Tamarix nilotica flowers. A total of 39 constituents including tannins, flavonoids, and phenolic acids, were tentatively identified. The in vitro cytotoxicity study revealed significant cytotoxic action towards the hepatocyte-derived cellular carcinoma cell lines, human liver (Huh-7). However, further studies are necessary to correlate this activity to the identified compounds to demonstrate T.nilotica as a prospective drug candidate that inhibits cancer.