High Performance Liquid Chromatography-mass Spectrometry Analysis of High Antioxidant Australian Fruits with Antiproliferative Activity Against Cancer Cells

Abstract

Background: High antioxidant capacities have been linked to the treatment and prevention of several cancers. Recent reports have identified several native Australian fruits with high antioxidant capacities. Despite this, several of these species are yet to be tested for anticancer activity.

Materials and methods: Solvent extracts prepared from high antioxidant native Australian fruits were analyzed for antioxidant capacity by the di (phenyl)-(2,4,6-trinitrophenyl) iminoazanium free radical scavenging assay. Antiproliferative activities against CaCo2 and HeLa cancer cells were determined by a multicellular tumor spheroid-based cell proliferation assay. Toxicity was determined by Artemia franciscana bioassay.

Results: Methanolic extracts of all plant species displayed high antioxidant contents (equivalent to approximately 7-16 mg of vitamin C per gram of fruit extracted). Most aqueous extracts also contained relatively high antioxidant capacities. In contrast, the ethyl acetate, chloroform, and hexane extracts of most species (except lemon aspen and bush tomato) had lower antioxidant contents (below 1.5 mg of vitamin C equivalents per gram of plant material extracted). The antioxidant contents correlated with the ability of the extracts to inhibit proliferation of CaCo2 and HeLa cancer cell lines. The high antioxidant methanolic extracts of all species were potent inhibitors of cell proliferation. The methanolic lemon aspen extract was particularly effective, with IC50 values of 480 and 769 μg/mL against HeLa and CaCo2 cells, respectively. In contrast, the lower antioxidant ethyl acetate and hexane extracts (except the lemon aspen ethyl acetate extract) generally did not inhibit cancer cell proliferation or inhibited to only a minor degree. Indeed, most of the ethyl acetate and hexane extracts induced potent cell proliferation. The native tamarind ethyl acetate extract displayed low-moderate toxicity in the A. franciscana bioassay (LC50 values below 1000 μg/mL). All other extracts were nontoxic. A total of 145 unique mass signals were detected in the lemon aspen methanolic and aqueous extracts by nonbiased high-performance liquid chromatography-mass spectrometry analysis. Of these, 20 compounds were identified as being of particular interest due to their reported antioxidant and/or anticancer activities.

Conclusions: The lack of toxicity and antiproliferative activity of the high antioxidant plant extracts against HeLa and CaCo2 cancer cell lines indicates their potential in the treatment and prevention of some cancers.

Summary: Australian fruit extracts with high antioxidant contents were potent inhibitors of CaCo2 and HeLa carcinoma cell proliferationMethanolic lemon aspen extract was particularly potent, with IC50 values of 480 μg/mL (HeLa) and 769 μg/mL (CaCo2)High-performance liquid chromatography-mass spectrometry-quadrupole time-of-flight analysis highlighted and putatively identified 20 compounds in the antiproliferative lemon aspen extractsIn contrast, lower antioxidant content extracts stimulated carcinoma cell proliferationAll extracts with antiproliferative activity were nontoxic in the Artemia nauplii assay. Abbreviations used: DPPH: di (phenyl)- (2,4,6-trinitrophenyl) iminoazanium, HPLC: High-performance liquid chromatography, IC50: The concentration required to inhibit by 50%, LC50: The concentration required to achieve 50% mortality, MS: Mass spectrometry.

Keywords: Anticancer activity; Illawarra plum; bush tomato; desert lime; functional foods; lemon aspen; muntries; native tamarind.

Figure 1

Antiproliferative activity of plant extracts and untreated controls against HeLa cancer cells measured as percentages of the untreated control cells. 1: Muntries fruit; 2: Illawarra plum fruit; 3: Native tamarind fruit; 4: Lemon aspen fruit; 5: Desert lime fruit; 6: Bush tomato fruit; M: Methanolic extract; W: Water extract; E: Ethyl acetate extract; C: Chloroform extract; H: Hexane extract; NC: Negative control. Results are expressed as mean percentages ± standard error mean of at least triplicate determinations. *Results that are significantly different to the untreated control (P < 0.01)

Figure 2

Antiproliferative activity of plant extracts and untreated controls against CaCo2 cancer cell lines measured as percentages of the untreated control cells. 1: Muntries fruit; 2: Illawarra plum fruit; 3: Native tamarind fruit; 4: Lemon aspen fruit; 5: Desert lime fruit; 6: Bush tomato fruit; M: Methanolic extract; W: Water extract; E: Ethyl acetate extract; C: Chloroform extract; H: Hexane extract; NC: Negative control. Results are expressed as mean percentages ± standard error mean of at least triplicate determinations. *Results that are significantly different to the untreated control (P < 0.01)

Figure 3

The lethality of the undiluted fruit extracts and control (1000 μg/mL) toward Artemia nauplii. Blue and green bars represent 24 h and 48 h mortality, respectively. 1: Muntries; 2: Illawarra plum; 3: Native tamarind; 4: Lemon aspen; 5: Desert lime; 6: Bush tomato; M: Methanolic extract; W: Water extract; E: Ethyl acetate extract; C: Chloroform extract; H: Hexane extract; NC: Negative (seawater) control; PC: Positive control (1000 μg/ml potassium dichromate). All tests were performed in at least triplicate, and the results are expressed as mean ± standard error mean

Figure 4

(a) Positive and (b) negative ion reverse phase-high performance liquid chromatography-mass spectrometry total compound chromatograms of 2 μl injections of lemon aspen methanolic extract

Figure 5

(a) Positive and (b) negative ion reverse phase-high performance liquid chromatography-mass spectrometry total compound chromatograms of 2 μl injections of lemon aspen aqueous extract

Figure 6

Chemical structures of lemon aspen fruit compounds detected in the methanolic and aqueous extracts (a) hydroxyethyl salicylate; (b) ferulic acid; (c) diftalone; (d) gingerol; (e) ketorolac glucuronide; (f) rutin; (g) theophylline; (h) luteolin; (i) diosmin; (j) dihydrokaempferol; (k) ellagic acid; (l) trimethyl ellagic acid; (m) chlorogenic acid; (n) (1S,5R)-4-hydroxy-6,7-dioxabicyclo[3.2.1]oct-2-en-8-one; (o) cantharidin; (p) calamanene; (q) hydroxycalamanene (r) lauroyl sarcosinate; (s) queuine; (t) dihydroartemisinin

Figure

Ian Edwin Cock