1.
Introduction
Compounds of plant origin have been used for the treatment and prevention of various diseases since time immemorial, and now they remain extremely important [1]. Our previous investigations suggest numerous plant extracts and essential oils as preventive and treating agents for medicine and veterinary [2]–[5].
Scientists around the world are trying to reveal whether plant-derived antioxidants are cancer inhibitors or enhancers, their potential role in cancer treatment, and possible action mechanisms and targets in cancer cells. A huge number of investigations confirming the cytotoxic and potential anticancer activity of such plants are existing. Recent review articles cite hundreds of research data that state the positive effects of different plants in cancer treatment [6],[7]. In other investigations it was shown that plant-derived metabolites can influence as enhancers and modulators of the activity of chemotherapeutic drugs in some types of cancers, sometimes expressing synergistic effects [8]. These could be possible due to different mechanisms such as blocking and/or reversing the drug-resistance mechanisms [9],[10] reducing the chemoresistance by decreasing efflux proteins, and promoting apoptotic cell death [11]. There are also described other possible mechanisms of anticancer influence of the metabolites, extracted from different plant species including inhibition of carcinogen activity, tumorigenesis, cell proliferation, angiogenesis, and induction of cell death. These can be associated with the possible modulation of reactive oxygen species (ROS) production, inhibition of nuclear factor kappa B (NF-κB), down/upregulation of mitogen-activated protein kinase (MAPK) activation, and, eventually, the regulation of epigenetic change [12].
Armenian flora is very rich in plants with high biological activity, which could be considered for the development of different preparations with therapeutic values [13]. Many of these plants are possibly capable of being applied in the treatment and prevention of cancer, but there is an extremely low quantity of scientific information about this.
Therefore, this open field for research as well as the high rate of cancer evidence in Armenia [14],[15] forced us to investigate the antiproliferative and cytotoxic effects of extracts, obtained from plants represented in the Armenian flora in order to find prospective herbal species.
2.
Materials and methods
2.1. Chemicals and reagents
All applied chemicals and reagents were purchased from Sigma-Aldrich Co. Ltd.
2.2. Plant material
The plant materials were harvested from the Tavush region of Armenia (1400–2400 m above sea level) according to already established protocol [13]. Identification of plant materials was done at the Department of Botany and Mycology, Yerevan State University (YSU) (Armenia). Plant voucher specimens were deposited to the Herbarium of YSU and added to the collection with assigned voucher numbers (For details see Table 1).
Plant crude extracts were prepared by maceration technique using pure ethanol (96%) [13]. Ground plant materials (100 mg) were soaked with ethanol at 10: 1 solvent-to-sample ratio (v/w). The mixture was vortexed for one minute and left in an ultrasonic bath for 15 min and centrifuged at 1000 rpm for 5 min. The supernatant was transferred into a new tube and further, fresh solvent was added to precipitate at the same ratio, vortexed for one minute again and left in the ultrasonic bath for the second round. After the second centrifugation the supernatants were combined. Thus, 50 mg DW/mL crude ethanol extracts were prepared. The concentration of dissolved compounds/yield in 50 mg DW/mL extract was determined by drying 500 µL of extract and weighing each sample in three independent replicates (See Table 2).
2.3. Cell lines and culture
HeLa (human cervical carcinoma) and A549 (human lung adenocarcinoma) cells have been maintained in Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% Human serum, 1x Pen/Strep. Cells have been seeded in tissue culture-treated multi-well plates at a maximum density of 2 × 105 cells/cm2. The cells have been propagated at 37 °C in an atmosphere of 5% CO2 in a CO2 incubator (Biosan S-Bt Smart Biotherm).
2.4. MTT assay
The MTT test [16] was performed to assess the inhibition of growth of HeLa and A549 cells exposed for 4, 24, or 72 h to different concentrations (0.5, 0.25, and 0.125 mg DW/mL) of the test-plant extracts. Treatments were performed as three technical replicates. Three independent replicates of each treatment were also performed. Cytotoxicity was expressed as percent growth inhibition of cells exposed to tested plant extract compared to control cells treated with the appropriate volume of solvent only (1% ethanol in the final mixture), whose growth was regarded as 100%. Results were expressed as accumulated survival index (ASI), calculated as the sum of areas under survival curves for each exposure time for the same concentration range (0.125, 0.250 and 0.5 mg DW/mL).
2.5. DPPH free radical scavenging assay
The antioxidant potential of tested extracts was evaluated spectrophotometrically, using DPPH (1-diphenyl-2-picrylhydrazyl) radicals according to the procedure described before [16]. The stock solution of DPPH was prepared prior to measurements; DPPH with ethanol until the absorbance reached 0.9 ± 0.02 at λ = 515 nm. Measurements of absorbance were carried out in 48-well plates using SPECTRO star Nano microplate reader (BMG Labtech, Germany). The stoichiometry values (n10) represent the number of oxidant molecules reduced by one molecule of antioxidant after 10 min of reaction were determined at room temperature. Regression coefficient, which was defined as the tangent of the line representing the relationship between the amount of scavenged DPPH (µg) of a radical scavenger and the quantity of the tested antioxidant extract present (µg) in the mixture after 10 min of reaction (n10) [17].
2.6. Determination of total phenolic content
The total phenolic content of plant extracts was measured exploiting the Folin & Ciocalteu's phenol reagent (FC) employing a calibration curve of gallic acid (GA) (0–250 µg/mL) using a UV-Vis spectrophotometer (Genesys 10S, Thermo Scientific, USA) [4].
2.7. Statistical analysis
All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc., USA). A p-value of less than 0.05 was considered significant. All results are presented as means ± SD (standard deviation). An unpaired Student's t-test (p ≤ 0.05) was used to evaluate antioxidant activity in the chemical (DPPH) test.
3.
Results and discussion
There is a high biodiversity of flora in the area of the Republic of Armenia which includes herbal species widely used in folk medicine [13],[18]–[21]. This diversity can also be a source of plant products with potential anticancer properties. During this investigation, we screened alcoholic extracts of 10 herbal species for their cytotoxic properties on cancer cell lines of different origin in order to select promising plant samples for further more targeted and comprehensive studies (See Table 1). The initial selection of herbal species was based on their uses in folk medicine which could indicate the possible anticancer activities according to traditional medical handbooks [19],[20].
At the initial stage of the study the effect of ethanol extracts of 10 herbal species on the growth of HeLa cells, which is one of the most common cancer cell lines, was evaluated. It is important to point out that only low concentrations of the extracts were used (ranging from 0.125–0.5 mg DW/mL) in order to find herbal species with strong cytotoxic properties. The extraction yield and concentration of dissolved compounds in 50 mg DW/mL extract are presented in Table 2.
Based on obtained data, five of the tested herbal extracts showed strong growth-inhibiting activity on HeLa cells (Figure 1). These plants are A. smirnovii, H. alpestre, I. helenium, C. pallasii (flowers with leaves) and R. canescens. For estimation of the overall cytotoxic properties of different herbal extracts accumulated survival index (ASI) parameters were calculated, which are defined as the sum of areas under survival curves determined for individual treatments for the same concentration range. ASI values are allowing to compare cell growth inhibiting effect of different samples. Obtained ASI parameters clearly indicated the high cytotoxic properties of these five extracts compared to the reminded samples (See Figure 1).
Further comparisons of the growth-inhibiting effectiveness of mentioned five samples based on higher cytotoxic effects at lower concentrations and short exposure times allowed the selection most promising three samples for more comprehensive analysis. These plant samples are A. smirnovii, H. alpestre, and I. helenium. The extracts from these plants or their relative species within the same genus are reported to possess considerable cytotoxic properties according to literature data as well. For instance, the strong cytotoxic activity of H. alpestre alcoholic extracts was revealed in our previous research work [5]. During the evaluation of its neuroprotective properties on microglial BV-2 cells we revealed the strong cytotoxic properties of this plant extract. This led to further evaluation of this plant extract as a source of cytotoxic agents. There was a lack of other literature reports about the cytotoxic influence of H. alpestre extract. However many of the species from the Hypericum genus were known to possess high cytotoxicity on different cancer cell lines [22]. For instance, considerable cytotoxicity of H. retusum flower methanol extracts on HeLa and NRK-52E cell lines was shown [23]. Literature reports about cytotoxic properties of I. helenium extracts mainly refer to its root extracts [24],[25]. For example, a highly selective cytotoxicity of I. helenium root extract on different tumor cell lines (HT29, MCF-7, Capan-2 and G1) was reported by Dorn et al. [26]. However, some data is also available about the cytotoxic properties of aerial parts of this plant as well. Aqueous extracts of I. helenium aerial parts were reported to exhibiting considerable cytotoxicity in a human U-87 MG glioblastoma cell line [27]. There was a lack of literature data about cytotoxic properties of A. smirnovii, however, high cytotoxic activity of the other species within a genus was reported (A. mollis, A. vulgaris, etc.) against different cancer cell lines, including breast, ovarian and cervical carcinoma [28]–[30].
Further, growth-inhibiting properties of selected three herbal extracts on human lung adenocarcinoma (A549) cells were explored. According to obtained data, only A. smirnovii and H. alpestre ethanol extracts showed strong cytotoxicity on A549 cells, whereas I. helenium extracted showed only slight growth inhibiting properties (Figure 2). ASI parameters also clearly point out the higher cytotoxic properties of A. smirnovii and H. alpestre extracts compared to extract of I. helenium (See Figure 2). It was important to mention, that exposure time had no significant impact on the growth-inhibiting the effectiveness of extracts of A. smirnovii and H. alpestre. Moreover, the A. smirnovii and H. alpestre extracts exhibit strong cytotoxicity even at the lowest tested concentration (0.125 mg DW/mL).
Antioxidant/pro-oxidant properties of the plant extracts can play an important role in the consent of possible anticancer properties. Therefore, for the assessment and selection of the most potent plants with possible anticancer properties their antioxidant properties were explored through DPPH colorimetric method.
In particular, the DPPH stoichiometry values of the test extracts were determined, which describe the number of oxidant molecules reduced by one molecule of antioxidant after 10 min of reaction (n10) (Figure 3a,b). According to obtained data, A. smirnovii ethanol extract possessed the highest radical scavenging activity followed by H. alpestre. I. helenium possessed low radical-scavenging activity compared to other plant samples. In our previous research work metal chelating, hydrogen peroxide reducing and DPPH reducing the activity of methanol extract of H. alpestre was shown [31]. High DPPH scavenging activity of A. smirnovii was reported for the first time. Though literature data confirms high radical scavenging activity of species within the genus Alchemilla, such as; Alchemilla vulgaris [28], Alchemilla. mollis [29], Alchemilla jumrukczalica [32].
Total content of phenolic compounds was determined in the selected plant extracts taking into account the importance of phenolics on biological activities of the extracts including antioxidant and cytotoxic properties [3],[4]. The highest total phenolic content was present in the extract of H. alpestre followed by A. smirnovii and I. helenium (Figure 3c). Generally total phenolic content of the plant extracts is in correlation with their radical scavenging activity [31],[33], however, A. smirnovii extract exhibited higher scavenging activity compared to H. alpestre extract. This phenomenon can be due to the high content of non-phenolic antioxidant compounds in A. smirnovii extract.
4.
Conclusions
Based on the screening of cytotoxicity of crude ethanol extracts of 10 herbal species on different cancer cell lines: two herbs with strong cytotoxicity were selected, which are A. smirnovii and H. alpestre. These herbs possessed high radical scavenging activity as well. Strong cytotoxic and antiradical activity of A. smirnovii was reported for the first time. We assumed that A. smirnovii and H. alpestre extracts could have promising anticancer potential and need further evaluation including elucidation of the metabolome, in vivo anticancer studies, etc. Although there is a lack of literature data about the cytotoxic properties of I. helenium areal part extracts, and it showed strong cytotoxic activity only on the HeLa cell line, we suggest that it can have great potential as a source of antitumor compounds and requires further investigation.
DownLoad:
