The Phytochemical Profile of the Petroleum Ether Extract of Purslane Leaves and Its Anticancer Effect on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-buta-4 None (NNK)-Induced Lung Cancer in Rats

Abstract

Lung cancer is a prevalent and very aggressive sickness that will likely claim 1.8 million lives by 2022, with an estimated 2.2 million additional cases expected worldwide. The goal of the current investigation was to determine whether petroleum ether extract of purslane leaf could be used to treat lung cancer induced by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-buta-4 none (NNK) in rats. In the in vitro extract recorded, promising anticancer effects in A540 cell lines with IC₅₀ were close to the reference drug, doxorubicin (14.3 and 13.8 μg/mL, respectively). A dose of 500 mg/kg/day orally for 20 weeks exhibited recovery effects on NNK-induced lung cancer with a good safety margin, where Intercellular Adhesion Molecule-1 (ICAM-1), the lung cancer biomarker, was significantly reduced by about 18.75% compared to cancer control. Purslane exhibited many anticancer mechanisms, including (i) anti-proliferation as a significant reduction in Ki67 level (20.42%), (ii) anti-angiogenesis as evident by a considerable decrease in Matrix metalloproteinase-9 (MMP-9) expression (79%), (iii) anti-inflammation as a remarked decline in Insulin-like growth factor 1 (IGF-1) expression (62%), (iv) pro-apoptotic effect as a significant activation in Forkhead box protein O1 (FOXO1) expression (262%), and (v) anti-oxidation as remarkable activation on antioxidant biomarkers either non-enzymatic or enzymatic concurrent with considerable depletion on oxidative stress biomarker, in comparison to cancer control. The histopathological examination revealed that Purslane extract showed markedly improved tissue structure and reduced pathological changes across all examined organs caused by NNK. The anti-lung cancer effect exhibited by the extract may be linked to the active ingredients of the extract that were characterized by LC-MS, such as α-linolenic acid, linoleic acid, palmitic acid, β-sitosterol, and alkaloids (berberine and magnoflorine).

Keywords: HPLC-QTOF/HR-MS/MS; Purslane leaf; lung cancer; phytochemical profile.

Figure 1

Portulaca oleracea picture: plant, leaf and flower, capsules, and seeds.

Figure 2

Effect of Purslane Pet. ether extract on A549 cells after 48 h. ^a: Significant change at p < 0.05 compared to control values.

Figure 3

Purslane leaf Pet. ether extracts decreased lung cancer biomarkers: (A), the relative weight of the lung, and (B), the ICAM-1 level. ICAM-1 (Intercellular Adhesion Molecule-1). Every value represents the mean of six replicates. The one-way ANOVA test was used to examine the data in order to compare means at p < 0.05. Those that have the same superscript letter in (A,B) do not differ substantially, while those that have different letters do at p ≤ 0.05.

Figure 4

Purslane leaf Pet. ether extract reduced the expression of the proliferative biomarker (Ki67) (A), angiogenic gene (MMP9) (B), and proinflammatory gene (IGF-1) (C) while inducing the expression of the apoptotic gene (FOXO1) (D). Every value represents the sum of six replicates. The one-way ANOVA test was used to examine the data in order to compare means at p < 0.05. Values in (A) that have the same superscript letter do not differ substantially from values that have different letters at p ≤ 0.05. Data in (B–D), ^a Compared to ve− control rats at p < 0.05. ^b Compared to rats with lung cancer at p < 0.05.

Figure 5

The antioxidant effect of Purslane leaf Pet. ether extracts in rats with lung cancer. Every value represents the sum of six replicates. The one-way ANOVA test was used to examine the data in order to compare means at p < 0.05. Values that have the same superscript letter do not differ substantially from values that have different letters at p ≤ 0.05, while those that have different letters do at p ≤ 0.05. (A) glutathione L reduced (GSH), (B) glutathione reductase (GR), (C) glutathione S-transferase (GST), (D) glutathione peroxidase (GPx), (E) catalase (CAT), (F) superoxide dismutase (SOD) and (G) lipid peroxidation biomarker (MDA).

Figure 6

Purslane leaf Pet. ether extract improved liver and renal performance; (A): total protein and its fractions (albumin and globulin), (B): liver enzymes (AST and ALT), and (C): renal functions (uric and urea). Every value represents the sum of six replicates. The one-way ANOVA test was used to examine the data in order to compare means at p < 0.05. Values that have the same superscript letter do not differ substantially from values that have different letters at p ≤ 0.05, while those that have different letters do at p ≤ 0.05.

Figure 7

Rat lung tissue sections stained with H&E in photomicrograph form. (A–D) represent the negative control and Purslane-treated groups, respectively, that displayed unremarkable histological changes with normal alveolar architecture and narrow interalveolar septa. After receiving progressively higher toxic doses of NNK, the NNK-treated group developed massive and severe interstitial macrophage infiltration within the alveolar spaces, apoptotic cells exhibiting cellular atypia, squamous cell metaplasia, and squamous dysplasia, as well as moderate to severe hyperplasia, adenoma, and adenocarcinoma (red arrows). A few portions under examination (E,F) exhibit alveolar hemorrhage. Reduced alveolar dysplasia and improved lung structures linked to slight hyperplasia in the blood vessel lining, mildly congested blood vessels, and mild macrophage infiltration were observed in the NNK and Purslane-treated group (yellow arrows) (G,H). (H&E (A,C,E,G) ×100, and (B,D,F,H) ×200).

Figure 8

Photomicrographs of a renal tissue section from the control group and Purslane ether extract groups (A–D), respectively, showing the normal histological renal structures. The NNK-treated group showed vacuolation of the renal tubules, severe congestion of renal blood capillaries, hypercellularity of the glomerular tuft, and renal hemorrhages (red arrows) (E,F). NNK and Purslane-treated group showing marked restoration of the normal renal histologic structures (G,H) (H&E (A,C,E,G) ×100, and (B,D,F,H) ×200).

Figure 9

Photomicrographs of liver tissue sections from the control group and Purslane ether extract groups (A–D), respectively, showing the normal histological hepatic structures. NNK-treated group showing massive steatohepatitis (red arrows), multifocal apoptosis, necrosis, and inflammatory cell infiltration (E,F). NNK and Purslane-treated group showing marked restoration of the normal hepatic histologic structures (G,H) (H&E (A,C,E,G) ×100, and (B,D,F,H) ×200).

Figure 10

Representative HPLC-QTOF/HR-MS/MS total ion current chromatogram (TIC); (A) and base peak chromatograms (BPC); (B) of Purslane leaves Pet. ether extract were analyzed using (−ve) ionization mode. The chromatographic conditions are specified in Section 2 (“results and discussion” Section). Table 1 lists all peaks’ identities, Rt values, mass error, and MS/MS data.

Figure 11

Representative HPLC-QTOF/HR-MS/MS total ion current chromatogram (TIC); (A) base peak chromatograms (BPC); (B) Purslane leaf Pet. ether extracts were analyzed using (+ve) ionization mode. The chromatographic conditions are specified in Section 2 (“results and discussion” Section). Table 1 lists all peaks’ identities, Rt values, mass error, and MS/MS data.

Figure 12

The structure skeleton of isoquinoline alkaloids tentatively identified by HPLC-HR/QTOF-MS/MS from Pet. ether extract.

Figure 13

The structure skeleton of cyclodopa amide alkaloids.

Figure 14

The structure skeleton of sterols tentatively identified by HPLC-HR/QTOF-MS/MS from Pet. ether extract.

Scheme 1

The suggested pathways for fragmentation of magnoflorine.

Scheme 2

The suggested pathways for fragmentation of menisperine.

Scheme 3

The suggested pathways for fragmentation of protoberberine alkaloids have OCH3 at C-9 and C-10.

Scheme 4

The suggested pathway for fragmentation of colletine.

Scheme 5

The suggested pathway for fragmentation of tembetarine.

Scheme 6

The suggested pathways for fragmentation of amino alkaloids.