Abstract
Guaianolide is a type of naturally occurring sesquiterpene lactone compounds that have attracted attention due to their diverse biological properties. In this work, a guaiane-type sesquiterpene lactone identified as deoxycynaropicrin (compound 1) was isolated and reported for the first time from the leaves of Centaurothamnus maximus with two known flavonoid derivatives namely luteolin 6-O-methyl ether (compound 2) and quercetin 3-methyl ether 5-O-glucopyranoside (compound 3). The cytotoxic activity of all the three compounds was evaluated against the THP-1 human leukemia cell lines. Moreover, flow cytometry was employed to explore the cell cycle arrest and apoptosis induction for the active compound. We found that compound 1 (deoxycynaropicrin) exerted the highest cytotoxicity while compounds 2 and 3 showed no activity. Cell cycle analysis showed that compound 1 arrested the cells’ population at the G2/M phase. Furthermore, THP-1 cells treated with compound 1 exhibited a marked increase in the apoptotic cells compared to the control. Overall, this study showed that deoxycynaropicrin induces cytotoxicity against human leukemia cell lines and provided an important insight into its potential therapeutic effects against leukemia cells.
1 Introduction
Centaurothamnus is a Centaurea monotypic genus indigenous to the southwestern Arabian Peninsula’s mountains [1,2,3]. The genus of Centaurothamnus is represented by around 200 species, is highly restricted in Saudi Arabia and grows in rare places of cliffs and steep hillsides [4]. Centaurothamnus maximus Wagentz and Dittri (Asteraceae) (Figure 1) is one of the most important species in this genus that is characterized by a branched leafy shrub of about 1.5 m in height. The plant also possesses a densely white-tomentose stem having lanceolate-elliptic leaves arranged in an alternating manner. The flowers are in magenta with faint sweet odor and thistle-like shape with a size of 3–4 cm long situated at the end of the branches. It is a paleoendemic species that grows in Yemen and has not possessed any traditional usage. [1]. The phytochemical survey of C. maximus revealed the presence of guaianolides, edusamanolides, germacranolides and elemanolides [5,6,7,8,9]. Some flavonoids [10], acetylenes [11,12], oxygenated homoditerpenoids [13] and an aliphatic ester [14] have been reported previously. The present literature also reports on guaianolide sesquiterpene lactones chlorojanerin, janerin and cynaropicrin isolated from C. maximus [15]. As we know, the family Asteraceae is a good source of guaianolides. Most of the guaianolides are known for their antimicrobial, antitumor, anthelmintic, antischistosomal and contraceptive properties [16]. The wide spectrum of activities makes guaianolides the compounds of interest for the discovery of new leads, but their toxicity made some restrictions, and because of this, no drug has come to the market at present. Lately, however, the guaianolide thapsigargin and prodrugs have recently provided novel drug discovery possibilities [17]. This again makes guaianolides interesting synthetic and biosynthetic targets because the natural source is often limited and cannot be used as a sustainable source for isolating the compound on a large (kilogram) scale [18].
C. maximus and its various parts collected from Aqabaat Al-Makhwah, Saudi Arabia.
In the present investigation, a guaiane-type sesquiterpene lactone (deoxycynaropicrin) was isolated and reported for the first time along with two known flavonoid derivatives luteolin 6-O-methyl ether (compound 2) and quercetin 3-methyl ether 5-O-glucopyranoside (compound 3) from the leaves of C. maximus. The chemical structure of deoxycynaropicrin (compound 1) was established with the help of spectroscopic techniques including UV, IR, NMR and MS. In addition, we showed that deoxycynaropicrin exerted promising cytotoxic and apoptotic effects against THP-1 leukemic cells.
2 Materials and methods
2.1 Plant material
Leaves of C. maximus were collected from Aqabaat Al-Makhwah, after tunnel # 13, Kingdom of Saudi Arabia, in 2016 and identified by Field Taxonomist at the Pharmacognosy Department, College of Pharmacy, King Saud University. A voucher specimen (Voucher # 15024) was deposited at the Pharmacognosy Department, College of Pharmacy, King Saud University.
2.2 Extraction and isolation
The air-dried powdered leaves (750 g) of C. maximus were extracted with 96% ethanol at room temperature, filtered and concentrated under reduced pressure to afford 60 g of dried extracts of the leaves. The obtained dried extract was defatted using MeOH. The MeOH soluble part (50 g) was suspended in 40% H2O/EtOH and successively partitioned with n-hexane (n-Hex.), chloroform (CHCl3) and n-butanol (n-BuOH) to afford the corresponding fractions. The chloroform fraction (10 g) was chromatographed over a silica gel column, starting with CHCl3 as a mobile system, and gradually increasing polarity with MeOH. The eluted fractions were monitored with TLC and similar fractions were combined to end up with 12 main fractions (CH.1-12). Fraction CH.1, eluted with 5% MeOH/CHCl3, was purified using two successive chromatographic steps including CC (10% EtOAc/CHCl3 and Rp-18 CC (25% H2O/MeOH) to obtain compound-1 in the pure form. Compound 2 was precipitated from fraction CH.3 and also eluted with 5% MeOH/CHCl3 after Sephadex LH-20 CC purification with 10% H2O/MeOH as solvent system. Fraction CH.11 eluted with 30% MeOH/CHCl3 was passed through Sephadex LH-20 CC (10% H2O/MeOH) to afford pure compound 3.
2.3 Cell viability (MTT) assay
To test the cytotoxic effect of isolated compounds, an MTT assay was performed as previously described [19]. Before treatment, 5 × 104 THP-1 cells were plated per well in a 24-well plate. Thereafter, cells were treated with various concentrations of each compound (5, 2.5, 1.25 and 0.625 µg/mL), while wells without treatment were used as a control. Following 24 h of treatment, 0.01 mL of MTT was added to each well. Then, wells were incubated at 37°C in 5% CO2 for 4 h to MTT reduction. Isopropanol (1 mL) with acidified HCL was then added and mixed thoroughly to solubilize the formazan product. After 10 min of plate shaking, growth was measured through absorbance measurement with an ELISA plate reader at 570 nm wavelength. Using OriginPro 8.5 software, the IC50 values were calculated from the dose–response curve.
2.4 Cell cycle analysis
As previously documented [19], a flow cytometric study of cell cycle distribution based on DNA content was performed. In brief, THP-1 cells at 1 × 105 cells were cultured in 12-well plates in the presence of compound 1 (2.5 and 5 μg/mL) for 24 h. After treatment, the cells were washed, centrifuged and fixed with 70% ethanol at 4°C for 4 h. After fixing, the cells were washed, centrifuged and then resuspended in 500 μL phosphate-buffered saline (PBS) containing PI (50 μg/mL) and RNase A (100 μg/mL) and incubated for a further 30 min in the dark. Finally, the cells were analyzed by flow cytometry (Cytomics FC 500; Beckman Coulter, CA, USA).
2.5 Annexin V-FITC/PI apoptosis assay
The Annexin V-FITC/PI kit (Biolegend, USA) was used to perform the apoptosis detection for the most active compound (1) following the manufacturer’s guidelines. Briefly, treated and untreated cells were harvested, washed with PBS and resuspended with 100 µL of Annexin-binding buffer (1×). Then, cells were stained with both Annexin-FITC and PI (5 µL each) for 15 min in the dark. Then, 400 μL of binding buffer was added and the apoptotic cells were measured by a fluorescence-activated cell sorter analysis (Cytomics FC 500).
3 Results and discussion
The phytochemical study of C. maximus resulted in the identification and chemical characterization of three compounds (compounds 1–3) (Figure 2). The phytochemical study revealed that compound 1 belonged to a class of sesquiterpene lactone (NMR data, Table 1) and proved to be a therapeutically potent entity as far as human leukemia cell lines are concerned. Compound 2 (luteolin 6-O-methyl ether) and compound 3 (quercetin 3-methyl ether 5-O-glucopyranoside) belonged to the class of flavonoids and exhibited no response with human leukemia cell lines.
Chemical structure of the isolated compounds.
1H NMR and 13C NMR data of compound 1
| Position | 1H δ H multi, J/Hz | 13C δ C multi |
|---|---|---|
| 1 | 2.87 m | 45.1 CH |
| 2 | (a) 1.66 m | 36.9 CH2 |
| (b) 2.10 m | ||
| 3 | 4.46 t, 7.6 | 73.4 CH |
| 4 | — | 152.2 C |
| 5 | 2.75 tr, 9.2 | 51.1 CH |
| 6 | 4.19 q, 9.1 | 78.6 CH |
| 7 | 3.11 m | 47.4 CH |
| 8 | 5.00 m | 74.1 CH |
| 9 | (a) 2.29 dd, 4.7, 3.6 | 36.9 CH2 |
| (b) 2.61 dd, 14.6, 5.2 | ||
| 10 | — | 141.9 C |
| 11 | — | 137.5 C |
| 12 | — | 169.3 C |
| 13 | (a) 5.53 d, 3.0 | 122.5 CH2 |
| (b) 6.12 d, 3.3 | ||
| 14 | (a) 4.86 s | 118.0 CH2 |
| (b) 5.07 s | ||
| 15 | (a) 5.29 s | 113.2 CH2 |
| (b) 5.40 s | ||
| 16 | — | 166.4 C |
| 17 | — | 135.9 C |
| 18 | 1.92 s | 18.2 CH3 |
| 19 | (a) 5.62 s | 126.6 CH2 |
| (b) 6.12 s |
Compound 1 showed a molecular ion peak at m/z 330 calculated for C19H22O5 with eight unsaturation numbers. A characteristic absorption bands appeared at 3,393, 1,753 and 1,710 cm−1 in the IR spectrum assigned for free hydroxyl group, γ lactone and ester functionalities, respectively. 1H NMR showed a characteristic signals for guaianolide-type sesquiterpene esterified with acrylate moiety. The γ lactone ring was confirmed by a lactonic proton at 4.19 ppm and a multiple proton at 3.11 ppm assigned for protons at C6 and C7. A carbonyl carbon that appeared at 169.0 ppm in 13C NMR also confirmed the cyclic ester structure [20]. The rest of 1H NMR spectral data for compound 1 showed six methylenes; four of them attributed for exomethylene groups, each appeared as two signals at 5.53 (d, J = 3.0 Hz), 6.12 (d, J = 3.3 Hz); 5.07 (s), 4.86 (s), 5.29 (s), 5.54 (s), 5.62 (s) and 6.12 (s) ppm assigned for positions 13, 14, 15 and 19. The other two methylenes were assigned for positions 2 and 9, resonating at 1.66–2.61 ppm. A singlet olefenic methyl appeared at 1.92 for position 18. Moreover, the 1H NMR displayed two oxygenated downfield protons at 4.46 (t, J = 7.6) and 5.00 (m) assigned for CH at C3 and C8, respectively. 13C NMR and DEPT experiments showed a total of 19 carbon atoms deduced to one methyl, six methylene, six methines and six quaternary carbons. 2DHSQC experiment was also useful to connect each carbon to its corresponding proton through one bond length. The exact positions for the exomethylene were confirmed by the HMBC experiment to be at positions 4, 10, 11 and 17. Also, the oxygenated protons were assigned at carbons 3 and C8 through J 2 and J 3 HMBC cross-peak bond correlations from H3 4.64 to C1, C4 and C5 at 45.1, 152.2 and 51.1 ppm, respectively, and from H8 5.00 ppm to C6, C7 and C16 at δ c 78.6, 47.4 and 166.4. respectively. The last correlation (from H8 to C16) together with the correlation from olefinic methyl at δ c 1.92 to ester carbonyl at δ c 166.4 and from exomethylene protons at 5.62, 6.12 ppm (C19) to C16 and methyl 18 proved esterification position by acrylate moiety is at position 8. The aforementioned data confirmed that compound 1 identified as erguin previously isolated from Centaurea deflexa [21].
3.1 Compounds’ cytotoxic effects
Sesquiterpene lactones have long been suspected as potential anticancer agents and more compounds in this class may provide information on the chemical substituents that are important for the anticancer activity [21]. In particular, the reported antileukemic effects of these compounds supported the idea that these classes could be useful in the treatment of leukemia [22]. Hence, the antiproliferative activity of isolated compounds was tested against the THP-1 leukemic cell lines. Our results indicated that only compound 1 decreased the cell viability in a dose-dependent manner (Figure 3), while compounds 2 and 3 did not display any activity. The reported IC50 of compound 1 on the THP-1 cell line at 24 h was 7.5 μM (Table 2).
Effects of different concentrations of compound 1 on cell viability during a 24 h incubation period. MTT assay was employed to determine the cell viability after treatment. The experiment was carried out in triplicate and the results are reported as mean ± standard deviation (SD).
IC50 values of compounds isolated from C. maximus against THP-1 cells
| Compound | IC50 (µM) |
|---|---|
| 1 | 7.5 ± 0.1 |
| 2 | — |
| 3 | — |
| Doxorubicin | 0.73 ± 0.02 |
3.1.1 Compound 1 caused G2/M cell cycle arrest in THP-1 cells
A cell cycle assay was carried out to assess whether compound 1 treatment led to changes in the cell cycle progression. THP-1 cells treated with 7.5 or 15 µM of compound 1 showed a higher G2/M population (22.6 ± 1.1 and 33.9 ± 1.4%, respectively) compared with 18.7 ± 0.9% in the control (Figure 4). In addition, compound 1 treatment caused a concomitant decrease in the proportion of cells in the G1 phase of the cell cycle from control (54.0 ± 1.5%) to (50 ± 1.2% and 41.8 ± 1.2%). Hence, our data suggested that compound 1 induced cell cycle arrest at the G2/M phase (Figure 4). The results described here are also in line with the findings of Ahmed et al., who reported that the janerin compound isolated from the same species demonstrated the same effect against the same cells [19]. Since uncontrolled cell division is a hallmark feature of cancer cells, novel compounds that inhibit cell cycle progression have been sought in hopes of expanding available cancer-directed therapies [23]. In fact, several standard anticancer drugs that are isolated from plants, such as etoposide and Taxol, exerted their effects through cell cycle arrest at the G2/M phase [24].
Induction of the G2/M cell cycle arrest by compound 1 in THP-1 cells. (a) Cells were treated with compound at 2.5 and 5 µg/mL concentration for 24 h while untreated cells were served as control. (b) Bar graphs represent the cell phases. The data are presented as the mean ± SD, *p < 0.05 and **p < 0.01 vs group.
3.1.2 Compound 1 caused apoptosis in THP-1 cells
Apoptosis is a vital biological process that permits the elimination of damaged or abnormal cells. As a result, inducing apoptosis appears to be an attractive goal for killing tumor cells as well as a useful strategy for cancer treatment [25]. Therefore, the ability of compound 1 to induce apoptosis initiation was further examined by staining the cells with Annexin V-FITC/PI for the quantification of apoptotic cells using flow cytometry. As shown in Figure 5, compound 1 exposure at (7.5 and 15 µM) concentrations resulted in dose-dependent increments of early apoptotic cells population (4.2.0 ± 0.8 and 16.7 ± 1%) compared to control (1.8 ± 0.4%). There was also a significant increase in the late apoptotic population (8.5 ± 0.7 and 51.0 ± 1.2%). Around 11% of the cell population displayed a necrotic death when treated with a high dose (5 µg/mL) of compound 1. A similar effect against human U-937 leukemia cells was reported for guaianolides compounds (chlorohyssopifolins and Linichlorin) isolated from Centaurea hyssopifolia and Centaurea linifolia, respectively. Both compounds demonstrated a potent apoptotic effect, which provided an important insight into the cell death pathway induced by guaianolides compounds [26].
Compound 1 induced apoptosis in THP-1 cells. The cells were treated at 2.5 and 5 µg/mL followed by FITC/PI staining. (a) Quadrant charts show A1 (necrotic cells), A2 (late apoptotic cells), A3 (live cells) and A4 (early apoptotic cells). (b) Apoptosis percentages of untreated and treated cells are represented as bars. Values are represented by mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001 vs group.
4 Conclusions
In comparison to other types of sesquiterpene lactones, guaianolide-type sesquiterpene lactones have attracted more attention due to their more cytotoxic effect. Herein, deoxycynaropicrin was isolated from the C. maximus for the first time. We found that deoxycynaropicrin (compound 1) inhibited the proliferation of THP-1 leukemia cells via apoptosis induction and triggered cell cycle arrest at the G2/M phase. These in vitro findings suggested that deoxycynaropicrin had a therapeutic potential against human leukemia cells, which is needed to confirm it using the in vivo model.
Acknowledgments
The authors are thankful to the Researchers Supporting Project number (RSP-2021/132), King Saud University, Riyadh, Saudi Arabia, for funding this research work.
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Funding information: This research was funded by Researchers Supporting Project number (RSP-2021/132), King Saud University, Riyadh, Saudi Arabia.
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Author contributions: Conceptualization, A.S.A.; methodology, F.A.N., N.A.S., O.A.B. and S.M.M.; validation and data curation, A.G. and W.M.A.; writing – original draft preparation, F.A.N. and N.A.S.; writing – review and editing, N.A.S., F.A.N. and A.S.A.; resources: M.R.H.; funding acquisition, A.S.A. All authors have read and agreed to the published version of the manuscript.
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Conflict of interest: The authors declare no conflict of interest.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: All the data related to these findings are included in the MS.
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