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Article

Insights into HPLC-MS/MS Analysis, Antioxidant and Cytotoxic Activity of Astragalus fruticosus against Different Types of Cancer Cell Lines

by
Mohamed Fayez Dekinash
1,
Tarek M. Okda
2,*,
Ehab Kotb Elmahallawy
3,*,
Fathy Kandil El-Fiky
4,
Gamal Abd El Hay Omran
2,
Emil Svajdlenka
5,
Naief Dahran
6,
Manal F. El-Khadragy
7,
Wafa A. Al-Megrin
7 and
El Moataz Bellah Ali El Naggar
1
1
Department of Pharmacognosy, Faculty of Pharmacy, Damanhour University, Damanhour 22511, Egypt
2
Department of Biochemistry, Faculty of Pharmacy, Damanhour University, Damanhour 22511, Egypt
3
Department of Zoonoses, Faculty of Veterinary Medicine, Sohag University, Sohag 82524, Egypt
4
Department of Pharmacognosy, Faculty of Pharmacy, Delta University for Science and Technology, Mansoura 35511, Egypt
5
Department of Natural Drugs, Faculty of Pharmacy, Masaryk University, 60300 Brno, Czech Republic
6
Department of Anatomy, Faculty of Medicine, University of Jeddah, Jeddah 21959, Saudi Arabia
7
Department of biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2022, 15(11), 1406; https://doi.org/10.3390/ph15111406
Submission received: 10 October 2022 / Revised: 4 November 2022 / Accepted: 7 November 2022 / Published: 14 November 2022
(This article belongs to the Special Issue Liquid Chromatography in Analysis of Bioactive Compounds for Pharmaceuticals, Cosmetics, and Functional Food Interest)
Browse Figures
Versions Notes

Abstract

:
Plants from the genus Astragalus are gaining attention for their pharmacological importance. However, the information available regarding the HPLC–MS/MS chemical profile of A. fruticosus is inadequate. In this study, we performed HPLC–MS/MS analysis using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). We tentatively identified 11 compounds in the A. fruticosus methanolic extract, including five flavonoidal and six saponin glycosides. The extract showed moderate antioxidant activity with 21.05% reduction in DPPH UV absorption. The preliminary cytotoxic screening against seven human cancer cell lines using 100 μg/mL extract showed prominent cytotoxic potential against colorectal cancer HCT–116 with 3.368% cell viability. It also showed moderate cytotoxic potential against prostate (DU–145), ovarian (SKOV–3) and lung (A–549) cancer cell lines with cell viability of 14.25%, 16.02% and 27.24%, respectively. The IC50 of the total extract against HCT–116 and DU–145 cell lines were 7.81 μg/mL and 40.79 μg/mL, respectively. The observed cytotoxicity of the total methanolic extract from the leaves against colorectal cancer might facilitate future investigations on cytotoxic agent(s) for disease management.

1. Introduction

The genus Astragalus is the largest genus in the family Leguminosae (Fabaceae), with over 3000 species of annual and perennial small shrubs and herbs. It is one of the largest and most diverse genera among angiosperms [1]. Astragalus has been used as a medicinal plant for over 1000 years. Radix Astragali (A. membranaceus and A. mongholicus) is an ancient and well-known Chinese traditional herbal medicine that is used to enhance resistance against bacterial and viral infections, treat immunological disorders, stimulate the circulatory system, control excessive sweating, and as a diuretic, hepatoprotective and heart tonic [2]. The chemical composition of Astragalus uniformly consists of major active compounds, including polysaccharides, flavonoids (free or glycosidic forms) and saponins [3]. Astragalus contains both cycloartane and oleanane saponins with the latter being relatively rare [2]. The saponin-rich purified mixture isolated from A. corniculatus showed protective activity against the hamster myeloid graffi tumor [4]. The saponins extracted from Astragalus hamosus showed anticancer activity against breast carcinoma cell lines [2].
Using Astragalus-based Chinese medicine in combination with chemotherapy for treating colorectal and lung cancer increases the treatment efficacy, reduces the side effects and improves the patients’ quality of life [5]. Astragalus extracts also have other biological activities, including antioxidant [6], hepatoprotective [7] and immunomodulatory activities [8]. The saponins isolated from A. hamosus, A. kahiricus and A. peregrinus showed dose-related modulation of lymphocyte proliferation [9]. There are 37 species of Astragalus in Egypt as described by Tackholm [10], while Boulos recorded only 32 species [11]. To our knowledge, until early 2022, previous phytochemical and biological research on members of genus Astragalus in Egypt included the following 15 species: A. sieberi [12,13,14], A. hamosus [9], A. kahiricus [7,9,15], A. vogelii [16], A. eremophilus [16,17], A. bombycinus [18,19], A. peregrinus [8,19,20], A. annularis [21], A. trimestris [21], A. tomentosus [22,23,24], A. spinosus [25,26,27], A. tribuloides [28], A. trigonus [29,30,31], A. alexandrinus [29,32] and A. cremophilos [33]. A recent study discussed potential somatic embryogenesis and micropropagation of the endangered plant Astragalus fruticosus to ensure its conservation. The study also identified the presence of luteolin and kaempferol flavonoids, pyrogallol, protocatechuic acid, p–coumaric acid, chlorogenic acid, ferulic acid and ellagic acid by comparing the high-performance liquid chromatography-ultraviolet (HPLC–UV) chromatograms of the plant’s ethanolic extract and reference compounds. Astragalus fruticosus ethanolic extract showed α–glucosidase inhibitory activity (IC50 = 44.8 µg/mL) and exhibited cytotoxic activity against breast (MCF–7) and leukemia (HL–60) cell lines (IC50 of 28.3 µg/mL and 49 µg/mL, respectively) [34].
Notably, the combination of the HPLC chromatographic resolution and the structural information by MS and MS2 allows tentative identification of phytochemicals in plant extracts [35,36,37,38]. The information available regarding the HPLC–MS/MS chemical profiling of A. fruticosus and their activity on different cell lines is limited. Here we performed a preliminary cytotoxic screening of the A. fruticosus Forssk methanolic extract against a panel of seven cancer cell lines and determined its IC50 against the most sensitive cell lines. Additionally, we performed high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) analysis of the methanolic extract, which is ideal for analyzing phytochemicals, including saponins and flavonoidal glycosides, due to its high sensitivity and selectivity.

2. Results and Discussion

2.1. HPLC–MS/MS Identification of Compounds

Using HPLC–MS/MS analysis, we tentatively identified 11 compounds from Astragalus fruticosus. Figure 1 shows the extracted ion chromatogram of the seven compounds identified using ESI-Ve ionization mode in the tandem mass spectrometry, and their HPLC/MS–MS data and spectra are listed in Table 1. The HPLC/MS–MS data and spectra of compounds identified using atmospheric pressure chemical ionization (APCI)+Ve ionization mode in the MS–MS are listed in Table 2. The mass spectra for tentatively identified compounds using HPLC–MS/MS showing glycon daughter peaks are provided in the Supplementary Figures S1–S11.

2.1.1. Compounds Identified Using ESI Mode

The peak at Rt 13 and m/z 640 was identified as the (M–H)− of 5–hydroxy isomucronulatol–2′,5′–di–O–glucoside(C29H37O16). This was further confirmed by MS2, where the loss of one glucopyranosyl unit (M–162), two sugar units and ring A resulted in the base peak at m/z 477, glycon signal at m/z 316 and the daughter peak at m/z 520, respectively. The 5–hydroxy isomucronulatol–2′,5′–di–O–glucoside was previously identified as a flavonoid component in Radix Astragali [39], and its structure and fragments are illustrated in Figure 2.
The peak at Rt 17.1 and m/z 579 was identified as the (M–H)− of 2′,4′–trihydroxy–flavone–8–C–α–arabinopyranoside–7–O–β–glucopyranoside (C26H27O15)− that was previously isolated and fully characterized from A. bombycinus [18]. The MS2 spectrum showed a distinctive base peak at m/z 284.8 representing the genin compartment due to the loss of the sugar units (M–(162) glucopyranosyl–(132) arabinopyranosyl)−. The daughter peaks at m/z 447 (M–132)− and m/z 429 (M–132–18)− were due to the loss of the arabinopyranosyl moiety. The loss of a glucose caused the daughter peak at m/z 417. Figure 3 shows the chemical structure and fragmentation of 2′,4′–trihydroxy–flavone–8–C–α–arabinopyranoside–7–O–β–glucopyranoside.
The compound eluted at Rt 17.7 and m/z 737.5 using ESI -Ve was identified as the (M–H)– of kaempferol–3–O–α–L– rhamnopyranosyl–(1→2)–[6–O–(3–hydroxy–3–methyl– glutaryl)–β–d–galactopyranoside] (KaeHMG), a flavonoidal glycoside that was previously identified in A. depressus [41], A. monospessulanus subsp. Illyricus [41] and A. gombiformis [40]. The MS2 spectrum demonstrated a peak at m/z 593 representing the loss of the terminal rhamnopyranose unit, which was previously documented in the MS2 spectrum for KaeHMG when the phenolic components of A. gombiformis were analyzed [40]. The peak at m/z 635 was due to the cleavage of the 3–hydroxy–3–methyl glutaryl moiety through McLafferty rearrangement at the carboxylic group attached to the sugar moiety. A peak at m/z 675 is probably due to loss of acetic acid from the 3–hydroxy–3–methyl glutaryl moiety and the formation of a stable tertiary carbocation attached to the methyl and hydroxyl groups. The aglycon kaempferol is observed at m/z 285. Figure 4 demonstrates the chemical structure and fragmentation of KaeHMG.
The peak eluted at Rt 18.9 and m/z 753.3 using the ESI - Ve mode was the (M–H)− (C33H37O20)− of quercetin–3–O–α–l–rhamnopyranosyl–(1→2)–[6–O–(3–hydroxy–3– methylglutaryl)–β–d–galactopyranoside] (QueHMG), which was confirmed by the MS2 spectrum where the base peak at m/z 609.2 was due to the loss of the rhamnopyranosyl terminal sugar unit. The same peak at m/z 609.1462 was observed in the MS2 spectrum of QueHMG, identified in A. monspessulanus using high resolution ESI–MS (HRESIMS) [41]. Another peak in the MS2 spectrum at m/z 651.1(M–C4H7O3)− was due to cleavage of the 3–hydroxy–3–methyl glutaryl moiety through McLafferty rearrangement at the carboxylic group attached to the sugar moiety. The genin was observed as a daughter peak at m/z 302 (M–rhamnopyranose– gluco–pyranose–H2O)−. Figure 5 shows the chemical structure and fragmentation of QueHMG.
The component eluted at Rt 20.7 and at m/z 931 using the ESI - Ve mode was identified as the (M–H)– of 3–O–[α–l–rhamnopyranosyl–(1→2)–β–d–xylopyranosyl]–24–O–β–d–glucopyranosyl–3β,6α,16β(24S),25–pentahydroxycycloartane, a cycloartane–type triglycoside saponin that was isolated and fully characterized from A. trojanus, and named as trojanoside C [42]. The MS2 spectrum showed a base peak at m/z 786 due to the loss of the terminal rhamnopyranosyl unit (M–146)−. The loss of isopropyl alcohol from the side chain created the daughter peak at m/z 869, and while that at m/z 562 was due to the loss of the terminal rhamnopyranose from C3, glucopyranose and isopropyl alcohol from C24 in the side chain (M–146–162–60)−. Figure 6 illustrates the chemical structure and fragmentation of trojanoside C.
The peak at Rt 21.3 and m/z 900 obtained using the ESI - Ve mode was identified as (M–H)− of 3–O–[α–l–Rhamnopyranosyl–(1→2)–β–d–xylopyranosyl]– 6–O–β–d– xylopyranosyl–20(R), 24(S)–epoxy–3β,6α,16β,25–tetrahydroxycycloartane, a cycloartane-type triglycoside saponin that was isolated and fully characterized from A. sieversianus [43] and A. trojanus [44], and named as Astrasieversianin XV. The MS2 spectrum demonstrated daughter peaks at m/z 755 and m/z 837 due to the loss of the terminal rhamnopyranosyl unit and isopropyl alcohol from the side chain, respectively. The peak at m/z 798 (M–C6H14O)− was created after cleavage of the side chain, resulting in a stable carbocation on the C20 carboxylic carbon. Figure 7 shows the chemical structure and fragmentation of Astrasieversianin XV.
One of the main components eluted at Rt 22.5, m/z 751.5 using ESI - Ve mode was identified as (M–H)− of 7–Methylkaempferol–3–O–α–l–rhamnopyranosyl–(1→2)–[6–O– (3–hydroxy–3–methylglutaryl)–β–d–galactopyranoside] (MethKaeHMG). The MS2 showed a base peak at m/z 607 due to the loss of the rhamnopyranosyl unit. The peak at m/z 689 might arise from the loss of one methoxy and two hydroxyl groups from the flavonoidal nucleus. Another fragment at m/z 650 (M–C4H7O3)− emerged from cleavage of the 3–hydroxy–3–methyl glutaryl moiety through McLafferty rearrangement at the carboxylic group attached to the sugar moiety. The aglycon peak of 7–methoxy kaempferol was observed at m/z 299. The peak at m/z 607 in the MS2 spectrum of MethKaeHMG was previously observed while studying the flavonoidal constituents of A. gombiformis [40]. Figure 8 provides the chemical structure and fragmentation of MethKaeHMG.

2.1.2. Compounds Tentatively Identified Using APCI Mode

The two main components eluted at Rt 20.9 and 22.2 at m/z 679 were identified as (M+H)+ of kahiricoside III and IV, respectively. Both are cycloartane-type saponin glycosides attached to acetylated glucose, where the acetyl group is attached to C2 in kahiricoside III and C6 in kahiricoside IV. They were previously isolated and fully characterized from A. kahiricus where the (M+H)+ was determined by high resolution fast atom bombardment (HRFAB)–MS at m/z 679.4412 for C38H62O10. Kahiricoside III was eluted first, followed by kahiricoside IV using C18 reversed-phase HPLC and methanol–water elution [15]. The MS2 spectrum indicated daughter peaks at m/z 661 and m/z 452 due to the loss of water and the acetylated glucose moiety with one water molecule, respectively. Those at m/z 643 and m/z 435 were due to the loss of two water molecules and the acetylated glucose compartment and two water molecules, respectively. Figure 9 shows the chemical structure and fragmentation of kahiricoside III and IV.
The peak eluted at Rt 34.6 and m/z 579 using APCI + Ve mode was identified as (M+H)+ of deacetyl tomentoside I, which was previously isolated and fully characterized from the Egyptian A. tomentosus. The daughter peak at m/z 533 was observed earlier in the MS2 spectrum of deacetyl tomentoside I isolated from A. tomentosus, probably caused by loss of the ethoxy group C2H5O from the side chain. The peak at m/z 385 represents the genin compartment with the dissociation of the ethoxy group. Figure 10 shows the structure and fragmentation of deacetyl tomentoside I.
The peak at Rt 44.4 and m/z 577 using APCI + Ve mode was identified as the (M+H)+ of β–sitosterol–β–d–glucoside. In the MS2 spectrum, the peak at m/z 415 (genin) represents the cleavage of the glycosidic linkage and loss of the glucopyranosyl unit (M–162). Loss of C2H4 from the genin moiety’s side chain. The formation of a secondary carbocation in the side chain was also observed at m/z 387 (M–162–28)+. The loss of the sugar and C3H7 from the genin side chain probably resulted in the peak at m/z 355. β–sitosterol–β–d–glucoside was previously isolated from A. tomentosus [24], A. tanae [45], A. sieversianus [47] and A. altaicus [46]. Figure 11 illustrates the structure and fragmentation of β–sitosterol–β–d–glucoside.

2.2. Cytotoxic Activity

Several natural products can prevent or even treat various cancers. Medicinal plants have been used to treat cancer as they can prevent or delay cancer onset, improve the immune system and the physiological status (35). Table 3 summarizes the cytotoxic screening of A. fruticosus leaf methanolic extract on seven cancer cell lines using concentrations ranging from 10 μg/mL–100 μg/mL. Following the preliminary cytotoxic screening, we generated dose response curves for different concentrations (0.02, 0.2, 2, 20 and 200 μg/mL) of the extract against colorectal (HCT–116) and prostate (DU–145) cancer cells, the most sensitive cell lines, (Figure 12) with IC50 values of 7.81 μg/mL and 40.79 μg/mL, respectively. Meanwhile, the results for different concentrations of the methanolic extract against HCT–116 and DU–145 cells are represented in Supplementary Tables S1 and S2. At 10 μg/mL, the leaf methanolic extract was not cytotoxic against all the tested cancer cell lines, with the cell viability ranging from 96% to 102%. However, at 100 μg/mL, this extract exhibited prominent cytotoxicity against the colorectal cancer cells (HCT–116) with only 3.368% cell viability. It also showed relatively moderate cytotoxicity against prostate (DU–145), ovarian (SKOV–3) and lung (A–549) cancer cell lines with cell viability of 14.25%, 16.02% and 27.24%, respectively. The breast cancer (MCF–7) cells were the most resistant (54.1% cell viability), while the osteosarcoma (MG–63) and hepatocellular cancer (HepG2) cells exhibited cell viability of 39.09% and 37.13%, respectively.
These results are consistent with previous studies on the Astragalus species in Egypt where the ethyl acetate fraction of the A. sieberie whole plant methanolic extract showed higher cytotoxicity against HCT–116 cells than MCF–7 cells with IC50 32.2 and 69.6 µg/mL, respectively [13]. Moreover, compounds isolated from A. spinosus roots [48] and A. kahiricus aerial parts exhibited cytotoxicity against the ovarian cancer cell line (A2780) with IC50 ranging from 16–47 µg/mL [15]. The methanolic extract of A. fruticosus showed dose dependent cytotoxicity against the two most sensitive cell lines, HCT–116 (colorectal) and DU–145 (prostate) cancer cells. The extract showed moderate cytotoxic activity against DU–145 cells (IC50 = 40.79 µg/mL), while that against HCT–116 cell line was significant (IC50 = 7.81 µg/mL). Figure 12 represents the dose response curves for the methanolic extract of A. fruticosus against HCT–116 and DU–145 cell lines.

2.3. Antioxidant Activity

DPPH is a stable radical compound with an intense violet color that fades upon reaction with antioxidants [49]. We found 21.05% reduction in the absorbance of the DPPH radical, indicating weak to intermediate antioxidant capacity. The percentage scavenging of DPPH radical was then expressed as trolox equivalent antioxidant capacity (TEAC) that was calculated as approximately 2.51 μg/mL from the calibration curve. This shows that the percentage scavenging activity of 100 µg/mL of the extract was equivalent to the antioxidant potential of 2.31 μg/mL of trolox. Combination of natural products eliminates toxic side effects of chemotherapy and decreases colon cancer incidence [50]. This is consistent with studies showing low to intermediate antioxidant potential of some members of Astragalus growing in Egypt, such as A. sieberi [13] and A. bombycinus [18].

3. Materials and Methods

3.1. Plant Materials

The aerial parts of Astragalus fruticosus were collected during the flowering stage from Rashid, 40 km east of Alexandria, Egypt in March 2021 [31°23′07.7″ N, 30°25′13.5″ E]. The plant was kindly identified by Professor Sherif Sharawy, Professor of Plant Taxonomy, Faculty of Science, Ain Shams University. A voucher specimen (a.f.001) was deposited in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Damanhour University. After air–drying, 10 gm of the aerial parts were pulverized and extracted with 100 mL absolute methanol using the Soxhlet apparatus at 70 °C. The methanolic extract was evaporated to dryness using a rotary evaporator at 60 °C.

3.2. HPLC–ESI/APCI–MS/MS Analysis

HPLC separation was performed using the Eclipse XDB C18 column (50 × 2.1 mm, 1.8 μm) (Agilent, Santa Clara, California, USA) using an HPLC system (Agilent HP 1100, USA). The mobile phase was composed of (A) methanol with 0.05% formic acid and (B) water with 0.05% formic acid. The compounds were separated using gradient elution profile: 0 min, A:B 10:90; 36 min, A:B 100:0; 50 min, A:B 100:0. Post-run time, 16 min. Chromatography was performed at 30 °C with a flow rate of 0.3 mL/min using the Bruker daltonik mass spectrometer (Bremen, Germany) equipped with ESI and APCI interfaces. For ESI, the ionization was on negative ion mode with Turbo Spray source, scan type Q1 MS 50–1200 m/z, scan rate 2000 Da/s, CUR gas 25, temperature 450 °C, gas 1 50, gas 2 40 and ion spray voltage 4500 V. For APCI, the ionization was on positive ion mode; the settings for the nitrogen drying and nebulizer gas were 5 mL/min (325 °C) and 60 psi. The APCI temperature and the capillary amperage were investigated before optimizing other parameters. Data analysis was performed using LC/MSD Trap Software 5.3 (Bruker daltonik). The compounds were tentatively identified by constructing an in-house mass spectra (MS/MS2) library of various Astragalus compounds from literature and comparing their molecular weights and fragmentation (MS/MS2).

3.3. Cytotoxicity Assay

We used 10 and 100 μg/mL of the leaf methanolic extract in DMSO to perform the preliminary cytotoxic assay against seven cancer cell lines. The ovarian (SKOV-3) and colorectal (HCT–116) cancer cells were maintained in RPMI media, while the others (lung A–549, prostate DU–145, breast MCF–7, osteosarcoma MG–63 and hepatocellular HepG2 cancer cell lines) were cultured in DMEM media. Both were supplemented with 100 mg/mL of streptomycin, 100 units/mL penicillin and 10% of heat-inactivated fetal bovine serum in humidified, 5% (v/v) CO2 atmosphere at 37 °C [50]. Cell viability was assessed using the sulforhodamine B (SRB) assay [51,52]. In 96-well plates, 100 μL cell suspensions (5 × 103 cells) were incubated in complete media for 24 h. The cells were treated with 100 μL media containing various concentrations of plant extracts. After treating for 72 h, the cells were fixed by replacing the 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 five times with distilled water. Then, 70 μL SRB solution (0.4% w/v) was added and incubated at room temperature for ten min in the dark. The plates were washed thrice 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, and the absorbance was measured in triplicates at 540 nm using a BMG LABTECH®– FLUOstar Omega microplate reader (Ortenberg, Germany). All the cell lines were obtained from Nawah Scientific Inc., (Mokatam, Cairo, Egypt). After the preliminary screening, five different concentrations (0.02, 0.2, 2, 20 and 200 μg/mL) of the extract were tested for their cytotoxic activity against colorectal (HCT–116) and prostate (DU–145) cancer cell lines, the most sensitive cell lines. Dose response curves were generated, and the cytotoxic activity of the plant extract was expressed as IC50.

3.4. Antioxidant Activity of the Methanolic Extract

The antioxidant capacity of 100 μg/mL A. fruticosus methanolic extract was determined using DPPH assay based on the redox potential of DDPH (Sigma–Aldrich, St. Louis, MO, USA). We prepared 1 mM DPPH solution (0.394 mg/mL) in methanol and then diluted it 1:10 to obtain a 100 μM solution (Abs at 515 nm = 0.5–0.6). Then, 500 μL each of the test sample and 100 μM DPPH solution were mixed in a cuvette. The negative control contained 500 μL each of methanol and DPPH solution. Both solutions were incubated in the dark at room temperature for 15 min, and absorbance was read at 515 nm using methanol as blank. The results were expressed as the percentage reduction in the radical absorbance [18].
[Abs max (negative control) − (Abs sample + DPPH)/Abs max] × 100
The antioxidant potential of the extract was compared to trolox as a reference antioxidant agent, and the results were expressed as TEAC. We constructed the calibration curve between inhibition percentage and trolox concentrations (12.5–0.3 μg/mL). We obtained a high correlation coefficient value of 0.9915, which indicated good linearity. We calculated the TEAC of the extract by substituting the inhibition percentage of the tested extract in the regression equation of the calibration curve.

3.5. Statistical Analysis

Data were expressed as the mean ± SD. The results were calculated using one way analysis of variance followed by Tukey multiple comparisons test. GraphPad Prism software (version 5) was used for all statistical analyzes and creating graphs.

4. Conclusions

To the best of our knowledge, this study is the first HPLC–MS/MS chemical profiling of A. fruticosus that demonstrates the predominance of flavonoidal and cycloartane-type saponin glycosides. These results agree with previous studies showing that the genus Astragalus is rich in these compounds. The observed cytotoxic activity of the total methanolic extract against colorectal cancer cells might facilitate further research efforts to isolate cytotoxic agent(s) for disease management. Further studies are underway to isolate, characterize and evaluate the potential efficacy of these cytotoxic phytochemicals against colorectal cancer cells. We have also initiated tissue culture experiments to enhance the production of potential compounds from the endangered plant A. fruticosus.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph15111406/s1: Table S1. Cytotoxic activity of different concentrations of the methanolic extract against colorectal cancer cell line HCT–116; Table S2. Cytotoxic activity of different concentrations of the methanolic extract against prostate cancer cell line DU–145; Figures S1–S11. The mass spectra for tentatively identified compounds using HPLC–MS/MS showing glycon daughter peaks are provided in the Supplementary file.

Author Contributions

M.F.D., T.M.O., F.K.E.-F., G.A.E.H.O. and E.M.B.A.E.N. were involved in the conception of the idea and methodology design and performed data analysis and interpretation. E.K.E., E.S., N.D., M.F.E.-K. and W.A.A.-M. participated in the methodology design, sampling and laboratory work and data analysis. T.M.O. and E.K.E. contributed their scientific advice, prepared the manuscript for publication and completed revisions. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R23), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Institutional Review Board Statement

Permission to collect the plant samples for research purpose was granted from Pharmacognosy Department, Faculty of Pharmacy, Damanhour University. No other permissions were needed.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and the supplementary materials.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

HPLC: High performance liquid chromatography, MS/MS2: Tandem mass spectroscopy, ESI: Electrospray ionization, APCI: Atmospheric pressure chemical ionization, Cpd No.: Compound number, m/z: Mass to charge ratio, Rt: Retention time, M+: Molecular ion, DMSO: Dimethyl sulfoxide, SRB: Sulphorhodamine B, TCA: Trichloroacetic acid, μg/mL: microgram per milliliter, TRIS: tris(hydroxymethyl)aminomethane, RPMI: Roswell Park Memorial Institute, DPPH: 1,1-diphenyl-2-picryl-hydrazyl radical, QueHMG: quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-d-galactopyranoside], MethKaeHMG: 7-Methylkaempferol-3-O-α-l-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-d-galactopyranoside], KaeHMG: kaempferol-3-O-α-l-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-d-galactopyranoside].

References

  1. Rather, S.A.; Wei, Y.; Wang, J.; Zhao, L.; Chang, Z. Comparative study of floral developments and its systematic importance in the genus Astragalus L. and Oxytropis DC. (Fabaceae, Leguminosae: Papilionoideae). Biologia 2021, 76, 865–888. [Google Scholar] [CrossRef]
  2. Ionkova, I.; Shkondrov, A.; Krasteva, I.; Ionkov, T. Recent progress in phytochemistry, pharmacology and biotechnology of Astragalus saponins. Phytochem. Rev. 2014, 13, 343–374. [Google Scholar] [CrossRef]
  3. Rios, J.; Waterman, P. A review of the pharmacology and toxicology of Astragalus. Phytother. Res. 1997, 11, 411–418. [Google Scholar] [CrossRef]
  4. Krasteva, I.; Toshkova, R.; Nikolov, S. Protective effect of Astragalus corniculatus saponins against myeloid graffi tumour in hamsters. Phytother. Res. Int. J. Devoted Pharmacol. Toxicol. Eval. Nat. Prod. Deriv. 2004, 18, 255–257. [Google Scholar] [CrossRef]
  5. Lin, S.; An, X.; Guo, Y.; Gu, J.; Xie, T.; Wu, Q.; Sui, X. Meta-analysis of astragalus-containing traditional chinese medicine combined with chemotherapy for colorectal cancer: Efficacy and safety to tumor response. Front. Oncol. 2019, 9, 749. [Google Scholar] [CrossRef] [Green Version]
  6. Asgarpanah, J.; Motamed, S.M.; Farzaneh, A.; Ghanizadeh, B.; Tomraee, S. Antioxidant activity and total phenolic and flavonoid content of Astragalus squarrosus Bunge. Afr. J. Biotechnol. 2011, 10, 19176–19180. [Google Scholar] [CrossRef]
  7. Allam, R.M.; Selim, D.A.; Ghoneim, A.I.; Radwan, M.M.; Nofal, S.M.; Khalifa, A.E.; Sharaf, O.A.; Toaima, S.M.; Asaad, A.M.; El-Sebakhy, N.A. Hepatoprotective effects of Astragalus kahiricus root extract against ethanol-induced liver apoptosis in rats. Chin. J. Nat. Med. 2013, 11, 354–361. [Google Scholar] [CrossRef]
  8. Verotta, L.; Guerrini, M.; El-Sebakhy, N.; Asaad, A.M.; Toaima, S.M.; Abou-Sheer, M.E.; Luo, Y.-D.; Pezzuto, J.M. Cycloartane saponins from Astragalus peregrinus as modulators of lymphocyte proliferation. Fitoterapia 2001, 72, 894–905. [Google Scholar] [CrossRef]
  9. Verotta, L.; Guerrini, M.; El-Sebakhy, N.A.; Assad, A.M.; Toaima, S.M.; Radwan, M.M.; Luo, Y.-D.; Pezzuto, J.M. Cycloartane and oleanane saponins from Egyptian Astragalus spp. as modulators of lymphocyte proliferation. Planta Med. 2002, 68, 986–994. [Google Scholar] [CrossRef]
  10. Tackholm, V.; Boulos, L. Students’ Flora of Egypt, 2nd ed.; Cairo University: Cairo, Egypt, 1974; pp. 261–271. [Google Scholar]
  11. Boulos, L. Flora of Egypt; Al-Hadara Publishing: Cairo, Egypt, 1999; Volume 1, pp. 320–336. [Google Scholar] [CrossRef]
  12. El-Mawla, A.; Attia, A. Production of flavonoids in cell cultures of Astragalus sieberi DC. Bull. Pharm. Sci. Assiut 2002, 25, 79–83. [Google Scholar] [CrossRef] [Green Version]
  13. Salem, M.A.; Farid, M.M.; El-Shabrawy, M.; Mohammed, R.; Hussein, S.R.; Marzouk, M.M. Spectrometric analysis, chemical constituents and cytotoxic evaluation of Astragalus sieberi DC. (Fabaceae). Sci. Afr. 2020, 7, e00221. [Google Scholar] [CrossRef]
  14. Verotta, L.; Tatò, M.; El-Sebakhy, N.A.; Toaima, S.M. Cycloartane triterpene glycosides from Astragalus sieberi. Phytochemistry 1998, 48, 1403–1409. [Google Scholar] [CrossRef]
  15. Radwan, M.M.; El-Sebakhy, N.A.; Asaad, A.M.; Toaima, S.M.; Kingston, D.G. Kahiricosides II–V, cycloartane glycosides from an Egyptian collection of Astragalus kahiricus. Phytochemistry 2004, 65, 2909–2913. [Google Scholar] [CrossRef] [PubMed]
  16. Perrone, A.; Masullo, M.; Plaza, A.; Hamed, A.; Piacente, S. Flavone and flavonol glycosides from Astragalus eremophilus and Astragalus vogelii. Nat. Prod. Commun. 2009, 4, 487–490. [Google Scholar] [CrossRef] [Green Version]
  17. Perrone, A.; Masullo, M.; Bassarello, C.; Bloise, E.; Hamed, A.; Nigro, P.; Pizza, C.; Piacente, S. Unusual cycloartane glycosides from Astragalus eremophilus. Tetrahedron 2008, 64, 5061–5071. [Google Scholar] [CrossRef]
  18. Ibrahim, L.F.; Marzouk, M.M.; Hussein, S.R.; Kawashty, S.A.; Mahmoud, K.; Saleh, N.A. Flavonoid constituents and biological screening of Astragalus bombycinus Boiss. Nat. Prod. Res. 2013, 27, 386–393. [Google Scholar] [CrossRef]
  19. Hussein, S.R.; Marzouk, M.M.; Kawashty, S.A. Flavonoids as chemosystematic markers of Astragalus bombycinus Boiss. and Astragalus peregrinus Vahl growing in Egypt. Egypt. Pharm. J. 2018, 17, 67–70. [Google Scholar] [CrossRef]
  20. Abd El-Latif, R.; Shabana, M.; El-Gandour, A.; Mansour, R.; Sharaf, M. A new isoflavone from Astragalus peregrinus. Chem. Nat. Compd. 2003, 39, 536–537. [Google Scholar] [CrossRef]
  21. El-Hawiet, A.M.; Toaima, S.M.; Asaad, A.M.; Radwan, M.M.; El-Sebakhy, N.A. Chemical constituents from Astragalus annularis Forssk. and A. trimestris L., Fabaceae. Rev. Bras. De Farmacogn. 2010, 20, 860–865. [Google Scholar] [CrossRef]
  22. Radwan, M.M.; Farooq, A.; El-Sebakhy, N.A.; Asaad, A.M.; Toaima, S.M.; Kingston, D.G. Acetals of Three New Cycloartane-Type Saponins from Egyptian Collections of Astragalus tomentosus. J. Nat. Prod. 2004, 67, 487–490. [Google Scholar] [CrossRef]
  23. El-Sebakhy, N.A.; Harraz, F.M.; Abdallah, R.M.; Asaad, A.M.; Orsini, F.; Pelizzoni, F.; Sello, G.; Verotta, L. Cycloartane triterpene glycosides from Egyptian Astragalus species. Phytochemistry 1990, 29, 3271–3274. [Google Scholar] [CrossRef]
  24. Abdallah, R.; Ghazy, N.; Assad, A.; El-Sebakhy, A.; Pirillo, A. Constituents of the Egyptian Astragalus tomentosus Lam. Pharmazie 1994, 49, 377–378. [Google Scholar]
  25. Korany, M.; Moneeb, M.; Asaad, A.; El-Sebakhy, N.; El-Banna, A. Analysis of astragalosides I, II and IV in some Egyptian Astragalus species and Astragalus dietary supplements using high-performance liquid chromatography/evaporative light scattering detector and non-parametric regression. Phytochem. Anal. PCA 2020, 31, 594–605. [Google Scholar] [CrossRef] [PubMed]
  26. Shabana, M. New flavonol glycoside from Astragalus spinosus forssk. Egypt. J. Pharm. Sci. 2002, 43, 19–27. [Google Scholar]
  27. Aboul-Enein, A.M.; El-Ela, F.A.; Shalaby, E.A.; El-Shemy, H.A. Traditional medicinal plants research in Egypt: Studies of antioxidant and anticancer activities. J. Med. Plants Res. 2012, 6, 689–703. [Google Scholar] [CrossRef] [Green Version]
  28. El-Sebakhy, N.A.; Asaad, A.M.; Abdallah, R.M.; Toaima, S.M.; Verotta, L.; Orsini, F. Constituents of Egyptian Astragalus tribuloides Del. Nat. Prod. Sci. 2000, 6, 11–15. [Google Scholar]
  29. El-Sebakhy, N.A.; Asaad, A.M.; Abdallah, R.M.; Toaima, S.M.; Abdel-Kader, M.S.; Stermitz, F.R. Antimicrobial isoflavans from Astragalus species. Phytochemistry 1994, 36, 1387–1389. [Google Scholar] [CrossRef]
  30. Shaker, K.H.; Bernhardt, M.; Elgamal, M.H.A.; Seifert, K. Triterpenoid saponins from Astragalus trigonus. Z. Für Nat. C 2001, 56, 699–702. [Google Scholar] [CrossRef]
  31. Gariboldi, P.; Pelizzoni, F.; Tatò, M.; Verotta, L.; El-Sebakhy, N.; Asaad, A.M.; Abdallah, R.M.; Toaima, S.M. Cycloartane triterpene glycosides from Astragalus trigonus. Phytochemistry 1995, 40, 1755–1760. [Google Scholar] [CrossRef]
  32. Orsini, F.; Verotta, L.; Barboni, L.; El-Sebakhy, N.A.; Asaad, A.M.; Abdallah, R.M.; Toaima, S.M. Cycloartane triterpene glycosides from Astragalus alexandrinus. Phytochemistry 1994, 35, 745–749. [Google Scholar] [CrossRef]
  33. Makboul, M.A.; El-Shanawany, M.; Abdel-Baky, A. Study of the lipid and flavonoid contents of Astragalus cremophilos Boiss. growing in Egypt. Bull. Pharm. Sci. Assiut 1984, 7, 380–388. [Google Scholar] [CrossRef]
  34. Zayed, R.A.; El-Sayed, A.S.; Ismaeil, W.M. In vitro Micropropagation, biological activities and phenolic profile of Astragalus fruticosus Forssk. Asian J. Plant Sci. 2022, 21, 192–202. [Google Scholar] [CrossRef]
  35. Mendonça, S.C.; Simas, R.C.; Reis Simas, D.L.; Leitão, S.G.; Leitão, G.G. Mass spectrometry as a tool for the dereplication of saponins from Ampelozizyphus amazonicus Ducke bark and wood. Phytochem. Anal. 2021, 32, 262–282. [Google Scholar] [CrossRef] [PubMed]
  36. Moqbel, H.; El Hawary, S.S.E.D.; Sokkar, N.M.; El-Naggar, E.M.B.; El Boghdady, N.; El Halawany, A.M. HPLC-ESI-MS/MS characterization of phenolics in Prunus amygdalus, cultivar “umm alfahm” and its antioxidant and hepatoprotective activity. J. Food Meas. Charact. 2018, 12, 808–819. [Google Scholar] [CrossRef]
  37. Ibrahim, R.M.; El-Halawany, A.M.; Saleh, D.O.; Naggar, E.M.B.E.; El-Shabrawy, A.E.-R.O.; El-Hawary, S.S. HPLC-DAD-MS/MS profiling of phenolics from Securigera securidaca flowers and its anti-hyperglycemic and anti-hyperlipidemic activities. Rev. Bras. Farmacogn. 2015, 25, 134–141. [Google Scholar] [CrossRef] [Green Version]
  38. El Newehy, N.M.; Abd-Alhaseeb, M.M.; Omran, G.A.; Harraz, F.M.; Shawky, E. Comparative metabolomics reveal intraspecies variability in bioactive compounds of different cultivars of pomegranate fruit (Punica granatum L.) and their waste by-products. J. Sci. Food Agric. 2022, 102, 5891–5902. [Google Scholar] [CrossRef]
  39. Chu, C.; Qi, L.-W.; Liu, E.-H.; Li, B.; Gao, W.; Li, P. Radix Astragali (Astragalus): Latest advancements and trends in chemistry, analysis, pharmacology and pharmacokinetics. Curr. Org. Chem. 2010, 14, 1792–1807. [Google Scholar] [CrossRef]
  40. Montoro, P.; Teyeb, H.; Masullo, M.; Mari, A.; Douki, W.; Piacente, S. LC–ESI-MS quali-quantitative determination of phenolic constituents in different parts of wild and cultivated Astragalus gombiformis. J. Pharm. Biomed. Anal. 2013, 72, 89–98. [Google Scholar] [CrossRef]
  41. Shkondrov, A.M.; Krasteva, I.N. High resolution LC-MS/MS screening for secondary metabolites in bulgarian species of genus Astragalus L. Química Nova 2021, 44, 683–688. [Google Scholar] [CrossRef]
  42. Bedir, E.; Çalis, I.; Aquino, R.; Piacente, S.; Pizza, C. Secondary metabolites from the roots of Astragalus trojanus. J. Nat. Prod. 1999, 62, 563–568. [Google Scholar] [CrossRef]
  43. Li-Xian, G.; Xiano-Bing, H.; Yu-Qun, C. Astrasieversianins IX, XI and XV, cycloartane derived saponins from Astragalus sieversianus. Phytochemistry 1986, 25, 1437–1441. [Google Scholar] [CrossRef]
  44. Bedir, E.; Tatli, I.I.; Calis, I.; Khan, I.A. Trojanosides I—K: New cycloartane-type glycosides from the aerial parts of Astragalus trojanus. Chem. Pharm. Bull. 2001, 49, 1482–1486. [Google Scholar] [CrossRef]
  45. Kavtaradze, N.; Alaniya, M.; Masullo, M.; Cerulli, A.; Piacente, S. New Flavone Glycosides from Astragalus tanae Endemic to Georgia. Chem. Nat. Compd. 2020, 56, 70–74. [Google Scholar] [CrossRef]
  46. Yili, A.; Aisa, H.; Isaev, M. Betulinic acid and sterols from Astragalus altaicus. Chem. Nat. Compd. 2009, 45, 592–594. [Google Scholar] [CrossRef]
  47. Iskenderov, D.; Keneshov, B.; Isaev, M. Triterpene glycosides from Astragalus and their genins. LXXVI. Glycosides from A. sieversianus. Chem. Nat. Compd. 2008, 44, 319–323. [Google Scholar] [CrossRef]
  48. Radwan, M.M.; El-Sebakhy, N.A.; Asaad, A.M.; Toaima, S.M.; Kingston, D.G. Spinocoumarin I, a new coumarin Derivative from Astragalus spinosus Forssk. Nat. Prod. Commun. 2007, 2, 919–922. [Google Scholar] [CrossRef]
  49. Zhang, Y.; Wang, B.; Lu, F.; Wang, L.; Ding, Y.; Kang, X. Plant-derived antioxidants incorporated into active packaging intended for vegetables and fatty animal products: A review. Food Addit. Contam. Part A 2021, 38, 1237–1248. [Google Scholar] [CrossRef] [PubMed]
  50. Tarek, M.O.; Sary, K.A.; Mohamed, A.K.; Mohamed, M.A. Chemopreventive and anticancer activities of indomethacin and vitamin D combination on colorectal cancer induced by 1,2-dimethylhydrazine in rats. Biomed. Rep. 2021, 14, 27. [Google Scholar] [CrossRef]
  51. Allam, R.M.; Al-Abd, A.M.; Khedr, A.; Sharaf, O.A.; Nofal, S.M.; Khalifa, A.E.; Mosli, H.A.; Abdel-Naim, A.B. Fingolimod interrupts the cross talk between estrogen metabolism and sphingolipid metabolism within prostate cancer cells. Toxicol. Lett. 2018, 291, 77–85. [Google Scholar] [CrossRef]
  52. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.; Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. JNCI J. Natl. Cancer Inst. 1990, 82, 1107–1112. [Google Scholar] [CrossRef]
Pharmaceuticals 15 01406 g001 550
Figure 1. The extracted ions chromatogram of the identified compounds using ESI-Ve ionization mode in tandem mass spectrometry.
Figure 1. The extracted ions chromatogram of the identified compounds using ESI-Ve ionization mode in tandem mass spectrometry.
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Figure 2. The structure and fragmentation of 5–hydroxy isomucronulatol–2′,5′–di–O–glucoside.
Figure 2. The structure and fragmentation of 5–hydroxy isomucronulatol–2′,5′–di–O–glucoside.
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Figure 3. The structure and fragmentation of 2′,4′–trihydroxy–flavone–8–C–α–arabinopyranoside–7–O–β–glucopyranoside.
Figure 3. The structure and fragmentation of 2′,4′–trihydroxy–flavone–8–C–α–arabinopyranoside–7–O–β–glucopyranoside.
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Figure 4. The structure and fragments of KaeHMG.
Figure 4. The structure and fragments of KaeHMG.
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Figure 5. The structure and fragments of QueHMG.
Figure 5. The structure and fragments of QueHMG.
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Figure 6. The structure and fragments of trojanoside C.
Figure 6. The structure and fragments of trojanoside C.
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Figure 7. The structure and fragments of Astrasieversianin XV.
Figure 7. The structure and fragments of Astrasieversianin XV.
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Figure 8. The structure and fragments of MethKaeHMG.
Figure 8. The structure and fragments of MethKaeHMG.
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Figure 9. The structure and fragmentation of kahiricoside III and IV.
Figure 9. The structure and fragmentation of kahiricoside III and IV.
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Figure 10. The structure and fragmentation of deacetyl tomentoside I.
Figure 10. The structure and fragmentation of deacetyl tomentoside I.
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Figure 11. The structure and fragmentation of β–sitosterol–β–d–glucoside.
Figure 11. The structure and fragmentation of β–sitosterol–β–d–glucoside.
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Figure 12. Dose response curves for the methanolic extract of A. fruticosus against HCT–116 and DU–145 cell lines.
Figure 12. Dose response curves for the methanolic extract of A. fruticosus against HCT–116 and DU–145 cell lines.
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Table
Table 1. Compounds identified from A. fruticosus and their HPLC–MS/MS2 data using ESI-Ve ionization.
Table 1. Compounds identified from A. fruticosus and their HPLC–MS/MS2 data using ESI-Ve ionization.
Cpd No.RtMS1MS2 DataCompound Name
113.0640 (M-H)-519 (M-ring A-H)-
477 (M-glucopyranose-H)-
316 (a glycon) (M-2 x glucopyranose)-		5-hydroxy isomucronulatol-2/,5/-di-O-glucoside [39]
217.1579 (M-H)-447 (M-arabinopyranose)-
429 (M-arabinopyranose-H2O)-
284.8 (a glycon) (M-glucopyranose-arabinopyranose)-		2/,4/-trihydroxy-flavone-8-C-α-arabinopyranoside-7-O-β-glucopyranoside [18]
317.7737.5 (M-H)-675 (M- acetic acid)-
635 (M-C4H7O3)-Mclafferty
593 (M-rhamnopyranose)-
285 (a glycon) (Kaempferol-H)-		kaempferol-3-O-α-l-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-d-galactopyranoside] [40]
418.7753.3 (M-H)-651.1 (M-C4H7O3)-Mclafferty
609.2 (M-rhamnopyranose)-,
302.0 (a glycon) Quercetin-		Quercetin-3-O-α-l-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-d-galactopyranoside] [41]
520.7931 (M-H)-869 (M-isopropyl alcohol)-
786 (M-rhamnopyranose)-,
562
(M-rhamnose-glucose-isopropyl alcohol)-
440 (a glycon)
(M-rhamnose-glucose-H2O)-		Trojanoside C [42]
621.3900 (M-H)-837 (M-isopropyl alcohol)-
797 (M-C6H14O)-Side chain
755 (M-rhamnopyranose)-
456 (a glycon)
(M-Rhamnopyranose-2 xylose)-		Astrasieversianin XV [43,44]
722.5751.5 (M-H)-689 (M-OCH3-2OH)-
650 (M-C4H7O3)-
607 (M-rhamnopyranose)-
299.1 (a glycon) 7-methoxykaempferol-		7-Methylkaempferol-3-O-α-l-rhamnopyranosyl -(1→2)- [6-O- (3-hydroxy-3-methylglutaryl)-β-d-galactopyranoside] [40]
Table
Table 2. Compounds identified in A. fruticosus and their HPLC–MS/MS2 data using APCI + Ve ionization.
Table 2. Compounds identified in A. fruticosus and their HPLC–MS/MS2 data using APCI + Ve ionization.
Cpd No.RtMS1MS2 DataCompound Name
820.9679 (M+H)+661 (M-H2O)+
435 (M-acetylated glucose-2H2O)+		kahiricoside III [15]
922.2679 (M+H)+661 (M-H2O)+
643 (M-2H2O)+
453 (a glycon-H2O)+
(M-acetylated glucose-H2O)+
435 (M-acetylated glucose-2H2O)+		kahiricoside IV [15]
1034.6579.3 (M+H)+533 (M-C2H5O)+
385 (M-xylose-C2H5O)+		deacetyl tomentoside I [22]
1144.4577.5 (M+H)+415 (M-glucopyranose) + (a glycon)
387 (M-glucopyranose-C2H4)+
355 (M-glucopyranose-C3H8O)+		β-sitosterol-β-d-glucoside [45,46,47]
Table
Table 3. Summary for the preliminary cytotoxic screening of A. fruticosus leaf methanolic extract.
Table 3. Summary for the preliminary cytotoxic screening of A. fruticosus leaf methanolic extract.
Cell LineCell Viability %
10 µg/mL100 µg/mL
Colorectal cancer HCT-11676.31793.68
Prostate cancer DU-14595.09314.253
Ovarian cancer SKOV-396.028816.0264
Lung cancer A-54997.520227.2388
Hepatocellular carcinoma HepG299.997337.1369
Osteosarcoma MG-63101.83639.0968
Breast cancer MCF-799.030854.104
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Dekinash, M.F.; Okda, T.M.; Elmahallawy, E.K.; El-Fiky, F.K.; Omran, G.A.E.H.; Svajdlenka, E.; Dahran, N.; El-Khadragy, M.F.; Al-Megrin, W.A.; El Naggar, E.M.B.A. Insights into HPLC-MS/MS Analysis, Antioxidant and Cytotoxic Activity of Astragalus fruticosus against Different Types of Cancer Cell Lines. Pharmaceuticals 2022, 15, 1406. https://doi.org/10.3390/ph15111406

AMA Style

Dekinash MF, Okda TM, Elmahallawy EK, El-Fiky FK, Omran GAEH, Svajdlenka E, Dahran N, El-Khadragy MF, Al-Megrin WA, El Naggar EMBA. Insights into HPLC-MS/MS Analysis, Antioxidant and Cytotoxic Activity of Astragalus fruticosus against Different Types of Cancer Cell Lines. Pharmaceuticals. 2022; 15(11):1406. https://doi.org/10.3390/ph15111406

Chicago/Turabian Style

Dekinash, Mohamed Fayez, Tarek M. Okda, Ehab Kotb Elmahallawy, Fathy Kandil El-Fiky, Gamal Abd El Hay Omran, Emil Svajdlenka, Naief Dahran, Manal F. El-Khadragy, Wafa A. Al-Megrin, and El Moataz Bellah Ali El Naggar. 2022. "Insights into HPLC-MS/MS Analysis, Antioxidant and Cytotoxic Activity of Astragalus fruticosus against Different Types of Cancer Cell Lines" Pharmaceuticals 15, no. 11: 1406. https://doi.org/10.3390/ph15111406

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