Abstract
This study sought to evaluate the pharmacological activity of metabolites isolated from the dried and lyophilized ethanol extracts as well as other solvent fractions of the currently endangered Puya chilensis Molina (Chagual) by analyzing their effects on a human hepatocellular carcinoma (HCC) cell line. We identified several active metabolites from Chagual extracts and two, in particular, carnosol, were found in all the prepared fractions. In addition, Chagual exhibited considerable cytotoxicity against the cancer cell line used in this study, with a half-maximal inhibitory concentration (IC50) of 0.44 ± 0.11 and 0.27 ± 0.04 after a 72-hour treatment and, therefore, has the potential for further investigation as a source of candidate therapeutic agents.
Introduction
The Bromeliaceae family members have a high diversity of taxa in the neotropical region with 58 genera and 3172 species, and Puya is a large genus with close to 200 species.1,2 Puya species are present in the mountain regions of Central America, in the mid to high elevations of the Andes, and south to lower elevations of Central Chile.
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The Puya species have considerable morphological variations due to the diverse climatic conditions where they grow, which range from wet to arid habitats. In Chile, the genus Puya grows in the Mediterranean area, and Puya chilensis Molina (Chagual) is a characteristic plant of the stony ground near the region that stretches from Coquimbo to Valparaiso. The inflorescence is a pyramidal scape with numerous and reflexed bracts, which are bipinnate where the lower half is densely covered with large greenish yellow flowers.
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“Chagual” is the local traditional name of P. chilensis and its conservation status conferred by the International Union for Conservation of Nature (IUCN) criteria has qualified it as of “least concern (LC)”. However, Chagual has been endangered by the decimation of the native forest area, change in soil use, and changes in global climatic conditions. In addition, the cultural use of this plant as a food item
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in the mountain region has contributed to its endangered status. The dietary use of this plant involves cutting the hypocotyl and removing all the leaves, which are steamed, thinly cut, and consumed as a salad or used to replace onions in some recipes. In Chile, the only Puya species that is eaten as a salad is Puya chilensis (Schmeda).
Over the last years, the bioactivities of some Bromeliaceae species have been demonstrated such as the pineapple stem (Ananas comosus L., family Bromeliaceae), which has proteases that have shown antiproliferative and proapoptotic effects in colon carcinoma. 5 In addition, other Bromeliaceae genera have been reported to exhibit antioxidant, photoprotective, and antinociceptive effects.1,2,6 However, the specific bioactivity or phytochemical properties of the Chilean Puya are not fully known. Therefore, the aim of this study was to determine the bioactivity of some metabolites isolated from Chagual and their effect on human hepatocellular carcinoma (HCC).
Materials and Methods
Plant material and extraction
Fresh P chilensis [identified by Aly Valderrama] 2 stems were purchased from a local collector in Melipilla (Metropolitana, Region, Chile) who sells P chilensis as fresh salad. The plant material, stored at −20 °C, was cut into 2 portions and chopped; one portion was dried at room temperature until constant weight while the other was lyophilized. Both samples were macerated separately in 1 L of ethanol, diethyl ether, and ethyl acetate (analytical grade) [10% w/v] in the dark at room temperature with constant agitation for 7 days. Then, the total extract was concentrated using a rotary evaporator at 40 °C and stored at −20 °C until the analysis (Table 1).
Metabolites Isolated from the Extracts of Puya chilensis.
General experimental procedures
A 6490 triple quadrupole liquid chromatography/mass spectrometry (LC/MS) system equipped with an electrospray ionization (ESI) source (Agilent Technologies, Palo Alto, CA, USA) was used. All determinations were performed under negative ionization mode at a capillary voltage of 3000 V. Nitrogen was used as the nebulizer (35 psi) and drying gas at 10 L/min at a temperature of 300 °C. The PMT, fragmenter, and skimmer were set at 850, 100, and 60 V, respectively. A full scan mass spectrum was acquired from m/z 100 to 1300. Data acquisition and processing were carried out using MassHunter MS Optimizer software (Agilent Technologies, Palo Alto, CA, USA).
The separation was performed using a C18 column; 150 × 4.6 mm I.D.; 5 µm particle size (Agilent Technologies, Palo Alto, CA, USA). The mobile phase consisted of water with 0.1% formic acid (phase a) and acetonitrile with 0.1% formic acid (phase b) at a flow rate of 0.6 mL/min. The linear elution gradient was from 80% (a) to 60% (a) in 20 min. Each run was followed by a 5-minute wash with 100% (b) and an equilibration period of 11 min with 80% (a)/20% (b). The total run time for the analysis was 20 min. Then, 10 µL of the sample was injected and the peaks were assigned based on the mass of the standard compounds (Sigma Aldrich, Münich, Germany). 7
Metabolite identification was performed using the METLIN database according to the ionization patterns of triple quad MS/MS spectra obtained for each peak (https://metlin.scripps.edu/index.php, Scripps Center for Metabolomics). Databases analyze the match between reference spectrum and experimental data; identification regarding >95% of probability of match and retention times (polarity) was used to determine the plausibility of correct identification (double control: polarity in elution and mass spectrum) [Supplementary Figure S1].
Cell culture
Human HCC HepG2 cells (American Type Culture Collection, ATCC, HB-8065) were grown in a monolayer culture in Dulbecco's modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotic-antimycotic agents (all Gibco, NY, USA) at 37 °C in a humidified 5% CO2 incubator.
Cytotoxicity assay
HepG2 cells were seeded in a 96-well plate at an initial density of 5 × 103 cells/well. Twenty-four hours later, the cells were treated with either the control (Milli-Q water) or various concentrations of Chagual for 48 and 72 h. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the cytotoxicity, as previously described. 7 The absorbance of the dissolved formazan crystals was measured at 540 nm using a microplate reader (Tecan infinite® f50, Grodig, Austria).
Results
Isolation of compounds
Diverse metabolites were isolated from the different fractions analyzed, and at least 2 metabolites were found in all the fractions. Carnosol, a potent antitumor polyphenol,8,9 was confirmed as a metabolite by comparing its spectral peak to standard spectra in the databases. The second metabolite found in all the fractions was determined to be similar to quinic acid 10 by comparing its spectrum with a reference spectrum in the database, but additional confirmatory identification methods are needed because diverse metabolites have similar spectra. The other metabolites correctly identified were 3,4-dihydroxybenzoic acid (DHBA), 3,4,5-trihydroxybenzoic acid (gallic acid, GA), 11 shikimic acid, 10 hesperetin, 12 and 8-C-glycosylkaempferol. 13
Cytotoxicity assay
Exposure of tumor cells to increasing concentrations (0.5-1 mg/mL) of Chagual extracts resulted in a dose-dependent decrease in cell viability at 48 and 72 h (Figure 1). The in vitro antitumor activity analysis of ethanolic extracts of the lyophilized (Figure 1A and B) and dried (Figure 1C and D), Chagual plant material revealed that they were highly cytotoxic with a half-maximal inhibitory concentration (IC50) of 0.44 ± 0.11 and 0.27 ± 0.04, the latter only after a 72-hour treatment (Figure 1B and D, respectively).
Effect of Chagual on growth of human hepatocellular carcinoma (HCC, HepG2). HepG2 cells were treated with various concentrations (0.5-1 mg/mL) of lyophilized and dried ethanolic extracts of the Chagual for (A and C) 48 and (B and D) 72 h. Cell viability was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Insert contains half-maximal inhibitory concentration (IC50) values ± standard deviation (SD). Data are mean ± SD of 3 independent experiments each performed in triplicate.
Discussion and Conclusions
The compounds isolated in this study have been reported in other plants; however, it was interesting to discover their presence in the Puya genus. Carnosol is an ortho-diphenolic diterpene (Figure 1).14,15 Carnosol has been reported to possess activity against prostate, skin, breast, leukemia, and colon cancers.8,9,16,21 Shikimic acid is a key intermediate in the biosynthesis of numerous aromatic compounds in plants and microorganisms, and although quinic acid is structurally related to shikimic acid, it is not an essential intermediate in the formation of aromatic compounds (Figure 1). 10 Shikimic and quinic acid occur singly or together in most species, with a lesser concentration of quinic acid in monocotyledons. 10
DHBA is a derivative of hydroxybenzoic acid that reduces the frequency or rate of spontaneous or induced tumors independent of the mechanisms involved. The corresponding spectrum assigned to GA (3,4,5 trihydrobenzoic acid) shows a peak corresponding to the molecular ion in negative mode [M-H] − = 169 (100% abundance base peak). In addition, a peak at m/z 183 (roughly 26% of abundance) was observed, corresponding to an additional methyl group, which may have been formed during the ionization electrospray that formed this adduct. GA is a naturally occurring plant polyphenol isolated from water caltrop. 22 GA is well known for its notable natural and marked antioxidative, antiallergenic, antimutagenic, anticarcinogenic, antiviral, antibacterial, and anti-inflammatory activities.23,25 Previous studies have demonstrated that GA selectively induces apoptosis in certain tumor cell lines including HL-60RG, HeLa, dRLh 84, PLC/PRF/5, and KB cells.11,26 In addition, GA has a significant role in preventing malignant transformation and the development of cancer in vivo. 27
Currently, to the best of our knowledge, there are limited reports on the effects of GA on human HCC.28,29 Therefore, further studies are required to evaluate the biological function and roles of GA in HCC. Hesperetin, a glycoside of hesperidin, is the predominant flavonoid in lemons and oranges (drug bank). It has inhibitory effects against colon 30 and skin 31 carcinogenesis. In human skin carcinoma cells, hesperetin altered the mitogen-activated protein kinase (MAPK) signaling pathway by modulating the expression levels of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as increasing the expression level of proteins involved in the apoptotic pathway. 3
8-C-glycosylkaempferol are a large group of metabolites that consist of the kaempferol skeleton with modification at position 3 or 7. 4 8-C-glycosylkaempferol has gained importance because of its antitumor properties. Recently, it was discovered to have antiproliferative activity against several human cancer cells, as well as the ability to induce the intrinsic apoptosis pathway in MCF-7 cells. 31
In the present work, compounds tentatively identified in the extracts of P chilensis are different from other works. 3 We also present for the first-time information on the antitumor properties of Chagual.
Supplemental Material
sj-docx-1-npx-10.1177_1934578X221091671 - Supplemental material for Cytotoxic Effect of Puya chilensis Collected in Central Chile
Supplemental material, sj-docx-1-npx-10.1177_1934578X221091671 for Cytotoxic Effect of Puya chilensis Collected in Central Chile by César Echeverria-Echeverria, Aly Valderrama-Villarroel, Marcelo Ortega, Rodrigo A. Contreras, Gustavo E. Zúñiga, Leonor Alvarado-Soto and Rodrigo Ramírez-Tagle in Natural Product Communications
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors would like to thank the financial support of FONDECYT 11170840 (C.E.E.).
Author’s Contribution
Conceptualization, R.R.T. and C.E.E.; Methodology, M.O. and C.E.E.; Validation, G.Z., R.C., and C.E.E.; Formal Analysis, R.C. and C.E.E.; Investigation, R.R.T.; Resources, C.E.E.; Writing—Original Draft Preparation, R.R.T.; Writing—Review and Editing, A.V.V.; Visualization, C.E.E.; Supervision, R.R.T.; Project Administration, L.A.S.
Ethical Approval
Not applicable, because this article does not contain any studies with human or animal subjects.
Informed Consent
Not applicable, because this article does not contain any studies with human or animal subjects.
Trial Registration
Not applicable, because this article does not contain any clinical trials.
Supplemental material
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References
Supplementary Material
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