Abstract
This study presents the results of DNA interaction of 52 methanol and 51 dichloromethane crude plant extracts with herring sperm DNA evaluated in terms of absorbance reduction of the peak, following the HPLC method described by Pezzuto and his colleagues. The 103 plant extracts belong to the Asteraceae, Euphorbiaceae, Melastomataceae, Rubiaceae, and Solanaceae families and were collected in the Regional Natural Park Ucumarí (RNPU; Colombia). The highest percentages of DNA interaction within the six plant families studied were shown by the methanol extracts of Schistocarpha sinforosi. Cuatrec. (68.30%) Aspilla quinquenervis. Blake (50.74%), and Verbesina nudipes. Blake (43.20%), all members of the Asteraceae family, followed by the methanol extract of Deprea glabra. (Standl.) A.T. Hunziker (41.01%), a species of the Solanaceae family. The dichloromethane plant extracts studied displayed, a low percentage of DNA interaction.
Introduction
DNA, as a carrier of the genetic information, is the primary target for the interaction of many drugs, due to the possibility of interfering with duplication and transcription of this fundamental macromolecule (Tayeb et al., Citation2003). Studies of small molecules that react at specific sites along DNA strands as reactive models for nucleic acid interaction provide routes toward rational development of chemotherapeutic agents. There are several types of sites where small ligands can bind to DNA biopolymer; they can occur (i) between two base pairs (full intercalation), (ii) in the minor groove, (iii) in the major groove, and (iv) on the outside of the helix (Asadi et al., Citation2005; Ni et al., Citation2006).
Consequently, several experimental physical methods have been developed to search for compounds that interact with DNA. Some of these methods are based on UV-visible, fluorescence, infrared, as well as 1H and 31P NMR, electron spin resonance (ESR), and Raman spectroscopies, electrochemistry, piezogravimetry, viscosimetry, unwinding of supercoiled DNA, circular and linear dichroism, x-ray diffraction, differential scanning calorimetry, and high-performance liquid chromatography (HPLC), among others (Pezzuto et al., Citation1991; Maleev et al., Citation2003; Wang et al., Citation2003; Hirohama et al., Citation2005; Ni et al., Citation2006). Of these numerous approaches, electrochemical, UV spectrophotometric and fluorescence methods have been preferred, because the small molecule–DNA interaction may be experimentally monitored by changes in intensity and position of the respective electrochemical or spectroscopic peak responses (Maleev et al., Citation2003; Ni et al., Citation2006).
Studies on plant extracts have led to the discovery of drugs for the treatment of cancer, such as vinblastine, vincristine, taxol, etoposide, and so forth. There is still a great interest in detecting molecules from plant sources that can interact at a site in a specific way and with less toxicity to DNA than many of the natural, synthetic, or hemisynthetic bioactive agents used currently (Liscovitch & Lavie, Citation2002). To pursue this goal, the interaction of 52 methanol and 51 dichloromethane extracts from 52 Colombian plants were evaluated with herring sperm DNA following the HPLC method developed by Pezzuto et al. (Citation1991).
Materials and Methods
The methanol used was HPLC grade and purchased from Mallinckrodt (Phillipsburg, NJ, USA), and HPLC-grade water was obtained by a Barnstead E-pure system (Barnstead Thermolyne, Dubuque, IA, USA). For the assay, herring sperm DNA and vincristine sulfate were purchased from Sigma (St. Louis, MO, USA). A Hewlett Packard HP-1100 liquid chromatograph (Palo Alto, CA, USA) equipped with a diode-array detector (DAD), a 20 µL Rheodine manual injector, and the software HP Chemstation A. 06.01 was used. The column was a Hypersil ODS (4.6 × 250 mm, 5 µm; Agilent Technologies, Santa Clara, CA, USA).
Plant material
The plants were collected at different zones of the Regional Natural Park Ucumarí (RNPU; Colombia) on February 2000 and October 2001. They were classified by Dr. F.J. Roldán and are listed in . A voucher specimen of each species was deposited at the University of Antioquia Herbarium (Medellín, Colombia). The collected plant materials were processed and extracted according to the protocols described by Niño et al. (Citation2006). Phytochemical screening was performed according to the procedures described by Harborne (Citation1980).
Table 1. Plants collected in the Regional Natural Park Ucumarí (RNPU), percentage of DNA inhibition, and results of the methanol extracts on the phytochemical screening
DNA interaction assay
In the procedure proposed by Pezzuto et al. (Citation1991) and followed in this study; 50 µL of DNA solution (100 mg/L) was mixed with 50 µL of HPLC-grade water. After homogenization, 20 µL of this solution was injected into the HPLC instrument, the solvent system H2O-MeOH (80:20 v/v) was used for elution at a flow rate of 1 mL/min, and the absorbance registered at 250 nm. The procedure was performed in triplicate to determine DNA absorbance (A.DNA).
Then, 50 µL of DNA (100 mg/L) solution was mixed thoroughly with 50 µL of each plant extract solution (250 mg/L) to be analyzed, and 20 µL was injected into the HPLC instrument. The elution was performed with the same system and flow of the previous steps. The procedure was repeated three-times with the same plant extract to determine sample absorbance (A.SAMPLE). The above procedure was repeated, but using 50 µL of vincristine sulfate (100 mg/L) to get the percentage of DNA interaction for the positive control. The percentage of DNA interaction (% Int) was determined by applying the following formula: % Int = [(A.DNA - A.SAMPLE)/A.DNA] × 100.
Results and Discussion
The percentages of DNA interaction with the different plant extracts evaluated in this study are shown in . Of the 52 methanol extracts evaluated, 14 (26.92%) showed DNA interaction equal to or higher than 20.00%. The best results were shown by seven extracts of the Asteraceae family, and those of Schistocarpha sinforosi. Cuatrec. (68.30%) and Aspilla quinquenervis. Blake (50.74%) displayed the highest DNA-interaction. These DNA interaction percentages were even superior to the one of vincristine sulfate (50.00%). It is also important to mention that no literature reports exist on the ethnopharmacological bioactivity of these Asteraceae family species. In addition, some other methanol plant extracts that showed a reasonable DNA interaction in the families assayed were Deprea glabra. (41.01%, Solanaceae), Alchornea grandiflora. (28.95%, Euphorbiaceae), and Palicourea petiolaris. (25.00%, Rubiaceae).
From the 51 dichloromethane extracts assessed, only four (7.84%) showed reasonable DNA interaction: Cinchona pubescens. (28.18%) and Palicourea petiolaris. (21.67%), both species belonging to the Rubiaceae family, Mikania leiostachya. (24.73%, Asteraceae), and Croton magdalenenesis. (27.08%, Euphorbiaceae). Interesting, only Palicourea petiolaris. was able to show DNA-interaction activities in both extracts tested.
The main secondary metabolites detected in the methanol extracts through phytochemical screening were alkaloids, sterols, triterpenoids, saponins, and tannins (see ), and to these metabolites could be attributed the DNA interaction showed by the plant extracts in this study. These secondary metabolites can bind to DNA through intercalation and nonintercalation modes. Examples of naturally occurring small ligands that have been reported to intercalate with DNA are the alkaloids harman, norharman, harmine and its derivatives, both berberine and its dimers, ellipticine and its derivatives, among others (Berger, Citation2001; Frei et al., Citation2002; Cao et al., Citation2005; Qin et al., Citation2006). Some examples of small ligands that do not intercalate with DNA are berberine, burasaine (Kluza et al., Citation2003), and the agent TAS-103 (Ishida & Asao, Citation2002).
In addition, the wide variation in the binding mode of small ligands present on plant extract to DNA interaction can occur depending upon the following factors: (i) variation in [DNA]/[plant extract concentration] molar ratio (Tayeb et al., Citation2003), (ii) changes in the ionic strength (Kluza et al., Citation2003), and (iii) changes in the size and structure of the small ligands (Ni et al., Citation2006). Thus, some of these factors are responsible for the DNA interaction found in this study.
In conclusion, according to the method followed in this research, 32.69% of the collected plants showed a DNA interaction higher than 20.00%; it can thus be inferred that the flora from RNPU has great potential as a source of phytocompounds with possible pharmacological applications. It is necessary to continue with the isolation, identification, and evaluation of the secondary metabolites responsible for this DNA interaction among the more active plant extracts.
Acknowledgments
The authors would like to thank The Universidad Tecnológica de Pereira for financial support of the project and the Corporación Autónoma Regional de Risaralda (CARDER) for granting permission to access the plant collection.
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