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. 2022 Sep 29;12(10):921.
doi: 10.3390/metabo12100921.

Phragmanthera austroarabica A.G.Mill. and J.A.Nyberg Triggers Apoptosis in MDA-MB-231 Cells In Vitro and In Vivo Assays: Simultaneous Determination of Selected Constituents

Marwa S Goda  1   2 , Sameh S Elhady  3 , Mohamed S Nafie  4 , Hanin A Bogari  5 , Raina T Malatani  5 , Rawan H Hareeri  6 , Jihan M Badr  1 , Marwa S Donia  1
Affiliations

Phragmanthera austroarabica A.G.Mill. and J.A.Nyberg Triggers Apoptosis in MDA-MB-231 Cells In Vitro and In Vivo Assays: Simultaneous Determination of Selected Constituents

Marwa S Goda et al. Metabolites. .

Abstract

Phragmanthera austroarabica (Loranthaceae), a semi-parasitic plant, is well known for its high content of polyphenols that are responsible for its antioxidant and anti-inflammatory activities. Gallic acid, catechin, and methyl gallate are bioactive metabolites of common occurrence in the family of Loranthaceae. Herein, the concentrations of these bioactive metabolites were assessed using high-performance thin layer chromatography (HPTLC). Methyl gallate, catechin, and gallic acid were scanned at 280 nm. Their concentrations were assessed as 14.5, 6.5 and 43.6 mg/g of plant dry extract, respectively. Phragmanthera austroarabica extract as well as the three pure compounds were evaluated regarding the cytotoxic activity. The plant extract exhibited promising cytotoxic activity against MDA-MB-231 breast cells with the IC50 value of 19.8 μg/mL while the tested pure compounds displayed IC50 values in the range of 21.26-29.6 μg/mL. For apoptosis investigation, P. austroarabica induced apoptotic cell death by 111-fold change and necrosis by 9.31-fold change. It also activated the proapoptotic genes markers and inhibited the antiapoptotic gene, validating the apoptosis mechanism. Moreover, in vivo studies revealed a significant reduction in the breast tumor volume and weight in solid Ehrlich carcinoma (SEC) mice. The treatment of SEC mice with P. austroarabica extract improved both hematological and biochemical parameters with amelioration in the liver and kidney histopathology to near normal. Taken together, P. austroarabica extract exhibited promising anti-cancer activity through an apoptosis-induction.

Keywords: HPTLC; MDA-MB-231 cells; Phragmanthera austroarabica; apoptosis; drug discovery; sustainability of natural resources.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of previously isolated compounds from P. austroarabica; (1) gallic acid, (2) methyl gallate, (3) catechin, (4) catechin-4′-O-gallate, (5) quercetin, (6) pectolinarigenin, (7) dillenetin-3-O-glucoside, (8) emodin, (9) emodin-8-O-glucoside, (10) chrysophanic acid, (11) chrysophanic acid-8-O-glucoside, (12) β-sitosterol-3-O-glucoside, (13) lupeol, (14) ursolic acid.
Figure 2
Figure 2
HPTLC spectrum of a standard mixture of gallic acid, catechin, and methyl gallate scanned at λ = 280 nm.
Figure 3
Figure 3
(a) HPTLC chromatogram of 1 µg/band of gallic acid, catechin, and methyl gallate scanned at λ = 280 nm; (b) HPTLC chromatogram of 10 µg per band of P. austroarabica extract scanned at λ = 280 nm.
Figure 4
Figure 4
Flow cytometry analysis of MDA-MB-231 untreated and treated cells with P. austroarabica crude extract (IC50 = 19.8 µg/mL, 48 h). (A) Annexin V/PI staining for apoptosis/Necrosis assessment; (B) DNA content-cell cycle analysis. “Values are expressed as Mean ± SD of three independent values. ** (p ≤ 0.001) significantly different between treated and control using unpaired t-test in GraphPad prism”.
Figure 5
Figure 5
(A) Caspase 3/7 inhibitory activity in cancer MDA-MB-231 cells was treated with P. austroarabica crude extract (IC50 = 19.8 µg/mL, 48 h) using the “CellEvent® Caspase-3/7 Green Flow Cytometry kit”, where “L, viable cells; A, apoptotic cells; N, necrotic cells; and D, dead cells”; (B) Bar presentation for comparison of apoptotic cancer cells due to active caspases 3/7 of the tested extract. The data are expressed as the mean ± SEM of three independent experiments in triplicate. ** (p ≤ 0.001) significantly different between treated and control using unpaired t-test in GraphPad prism”.
Figure 6
Figure 6
Cell death through autophagy in untreated and treated MDA-MB-231 cells with P. austroarabica crude extract (IC50 = 19.8 µg/mL, 48 h) using the acridine orange lysosomal stain coupled with the flow cytometric analysis. Red: Negative control (untreated), Blue: treated cells.
Figure 7
Figure 7
Histopathological examinations of liver and kidney tissues in (A1,A2) Normal control; (B1,B2) SEC control; (C1,C2) P. austroarabica-treated SEC group. Portal area (PA); chronic inflammation (blue arrow); hydropic degeneration (Arrow heads). Normal glomeruli (Black arrows), urinary spaces, and tubules (Red arrows).
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