INTRODUCTION
For centuries, the species of the genus Achillea (Asteraceae) have been used in traditional medicine. Phytochemical investigations have revealed that Achillea species contain a broad range of highly bioactive compounds [1]. In medicine, current studies are focused on secondary metabolites as a source of new effective anticancer agents [2–4].
The plant assays are used for preliminary screening of the plant extracts for their cytotoxic activity: Allium cepa root tip bioassay is an adequate system for assessing mitotic repressant activity [5]. In vitro studies using different cancer cell lines are useful tools to investigate the mechanism of plants`extracts cytotoxic action [6–9]. Cytotoxic and pro-apoptotic effects of water extracts of Achillea species in vitro have been reported [10]. Nowadays, medicinal plants` chemical compounds are largely explored by humans. They are based on the therapeutic use of plants by indigenous people. Traditional medicines usually are prepared as whole water extracts of plants. Current studies have established different biological activity of cold and hot water extracts [11,12].
Achillea thracica is a critically endangered Bulgarian endemic species [13]. The aerial parts of A. thracica contain numerous secondary metabolites [14]. In modern society, a lot of valuable plant species are at risk of extinction. Ex situ conservation through the use of in vitro cultures may preserve the threatened plants [15,16]. In order to preserve A. thracica, a team from Sofia University provided a system for ex situ conservation through in vitro cultivation and ex vitro adaptation [17]. The chemical composition of plants depends on different factors. However, the constituents and properties of propagated plants should be studied.
The aim of this study was to evaluate the anticancer effects of water extracts of ex vitro established A. thracica Velen. grown in Bulgaria against fibrosarcoma cells.
MATERIALS AND METHODS
Water extracts
Ex vitro established plants were grown in the experimental field in Lozen mountain, Bulgaria [18]. Aerial parts of plants, cut about 30 cm from the top, were collected during the flowering period and dried at room temperature. Water extracts were prepared: 1) Hot extract (HE) - the air-dried and finely ground aerial parts were covered with boiling distilled water for 60 minutes. 2) Cold extract (CE) - the air-dried and finely ground aerial parts were placed in distilled water and left to stay for 24 hours at room temperature. Then, the aqueous extracts were filtered and evaporated at 50°C to obtain a dry extract.
Root elongation assay
The HEs were prepared at concentrations 8, 10, 12, 14, 16, 18 and 20 g/l, and the CEs - at concentrations 5, 10, 15, 20, 25 and 30 g/l using the corresponding dry extract. Seeds of Triticum aestivum L. were used. Five ml of each extract or distilled water as a control were applied to the seeds. The dishes were sealed and incubated at 25°C ± 1°C for 72 hours. The length of the roots of germinated seeds was measured. The percentage of root growth inhibition in relation to the control for each extract was determined. From the root growth curve, EC50 values were obtained: the effective concentration that decreased root growth about 50% when compared to the negative control group (distilled water, 100%) [19].
Allium cepa L.-test
Potential cytotoxicity and genotoxicity of water extracts were estimated using A. cepa L. (2n = 16) as a test object. The bulbs were kept for root germination in distilled water for 24 hours. Bulbs with new roots with a length of 1.5 cm were treated with HE and CE at concentrations ½ ??50 and ??50 of corresponding dry extract. Treatments were for 24 hours at 25°C ± 1°C. Temporary slide preparations were prepared [19]. The microscopic analysis included assessment of the mitotic index and aberrant cells.
In vitro cytotoxicity and pro-apoptotic activity of HE of A. thracica
Cell lines and culture conditions
The adherent HT1080 cell line fibrosarcoma cells were maintained in controlled environment: Dulbecco’s Modified Eagle Medium, supplemented by 10% Fetal calf serum at 37°C and 5% CO2.
Test-object and treatment
HT1080 cells were treated with dry extract from HE at concentrations of 50, 100, 200, 400 and 600 μg/ml for 48 hours. 5-Fluorouracil at a concentration of 50 μg/ml was used as a positive control. After treatment the cells were harvested from cell culture, and in vitro cytotoxicity and pro-apoptotic activity of A. thracica HE were evaluated.
In vitro cytotoxicity
Cells were stained with trypan blue dye and analyzed in a Bürker chamber. The cytotoxic effect of the extracts was evaluated by the change in the total number of cells, viability, and population growth rate [20]. The growth rate constant was calculated as follows:
where
? - growth rate constant;
N0 - number of cells at the start of the treatment;
N - number of cells after the treatment;
T - time of treatment.
Pro-apoptotic activity
HE-treated cells were harvested and fixed in Clarke’s fixative. The cell suspension was spread on a microscope slide and after drying was stained with Giemsa dye. Microscopic preparations were examined for the presence of cells undergoing apoptosis. As indicators of apoptosis, cell nuclei with morphological changes were used - pyknosis (irreversible condensation of chromatin causing the nuclei to decrease in size), karyorrhexis (destructive fragmentation of a pyknotic nucleus) and karyolysis (nuclear fading caused by the dissolution of chromatin) [21].
Data processing and statistics
Student’s t-test was performed with p ≤ 0.05 taken as a significance level.
RESULTS AND DISCUSSION
Influence on root growth of Triticum ?estivum L.
The potential antimitotic effect of ex vitro established A. thracica on root growth of T. ?estivum was evaluated. There are data about differences between extracts obtained at different temperatures [22]. Based on these observations, in the present study, we used HE and CE.
The results showed that both extracts caused similar effects on root growth (Fig. 1). An inhibitory effect on root growth as compared with controls was observed. Other studies also noticed that root elongation is a sensitive growth marker [23,24].
The Effective Concentration for 50% growth inhibition was estimated: EC50 = the effective concentration that decreased root growth by about 50% when compared to the negative control group [25,26]. EC50 value represents not only the degree of influence of extracts, but also usually is the first step in cytogenetic study of medicinal plants [27–29]. As can be seen from Figure 1, the HE exerted a stronger inhibitory effect on root growth in comparison with the CE. Respectively, the EC50 value of the hot water extract is 9.85 g/l and of the CE - 12.71 g/l. It can be speculated that the two extracts tested contain different bioactive compounds and/or different concentrations.
Allium cepa L.-test
Cytotoxic compounds could disturb the cell division [30]. Potential cytotoxicity of plant extracts was tested at concentration equal to ½ ??50 using A. cepa-test. The extracts tested showed a significant mitodepressive effect - the mitotic indices were extremely low (Table 1). In such a case it is not recommended to score chromosomal aberrations [26].
The decline of the mitotic index indicates the occurrence of a cytotoxic effect [31]. The findings of this study showed the possibility of use ex vitro adapted A. thracica as an anticancer drug.
 | Figure 1. Effect of A. thracica water extracts on root lenght of T. aestivum L. seeds. *p ≤ 0.05. [Click here to view] |
In vitro cytotoxicity and pro-apoptotic activity of HE of ex vitro established A. thracica on human fibrosarcoma cell line HT 1080
It is well known that conventional medicine uses different plant-derived compounds to treat cancer [24]. The secondary metabolites in medicinal plants are widely studied as new potential anticancer agents. Cytotoxic effects of different Achillea species have been reported [10].
Evaluation of the antimitotic effect of plant extracts on Allium root tips is a part of preliminary anticancer studies [5]. Positive results disclose the possibility to be used as an antineoplastic drug [32]. In vitro cell test systems are widely used to establish the specific properties of chemical compounds [33]. The human cell lines of pathogenic origin offer the opportunity to evaluate the therapeutic effectiveness of potential new drugs directly on cancer cells [34]. The results of the phytotoxicity test of A. thracica water extracts revealed stronger growth inhibitory effect of the HE. However, we tested the possible cytotoxicity of the HE on cancer cells in vitro. In the present study, human fibrosarcoma cell line HT 1080 was used as a test-object.
In vitro cytotoxicity effect on the cell population
The results of the treatment with HE of A. thracica on the total cell number, the cell viability and the population growth rate are summarized in Table 2. The cell number was reduced to a great degree after treatment with extracts at concentrations of 400 and 600 μg/ml - respectively 2.7 and 4.8 fold as compared to the untreated control. The extracts tested significantly inhibited the proliferation/viability of cells. The growth rate constant was lowered. The control cell population increased by 8.18% every hour. After treatment with extracts at concentrations of 400 and 600 μg/ml, this value was respectively 6.09% and 4.91% hourly.
Pro-apoptotic activity
The cancer cells can be eliminated by induction of apoptosis [35]. The morphological changes in the cell nucleus serve as signs of apoptosis [36–38]. The ability of studied extracts to induce apoptosis was assessed by the evaluation of nuclear morphology changes in Giemsa-stained cells. Three stages of the nucleus at the early stage of apoptosis were established - pyknosis, karyorrhexis and karyolysis.
 | Table 1. Effect of treatment with A. thracica water extracts at concentrations ½ ??50 and ??50 (for 24 hours) on root tip meristematic cells of A. cepa L. [Click here to view] |
 | Table 2. In vitro cytotoxicity effect of treatment with A. thracica hot water extract (for 48 hours) on human fibrosarcoma cell line HT 1080. [Click here to view] |
 | Table 3. Proapoptotic effect of treatment with A. thracica hot water extract (for 48 hours) on human fibrosarcoma cell line HT 1080. [Click here to view] |
Treatment with extracts increased significantly (three- to fivefold) the percent of the cells with nuclear morphology changes in comparison with the negative and the positive control (Table 3). It should be noted that pyknosis was established after treatment with all extracts. Treated with extract cells revealed also a sign of karyorrhexis. The nucleus alterations could be a reason for these results [38]. Karyolysis have been observed only in the treated cells and in the positive control. The frequency of induction of karyolysis is dose-dependent.
Many antitumor drugs are known to be effective only against certain tumor cells. Therefore, the first step in the investigation of new antitumor drugs is to establish their activity against tumor cells of different cell lines [39]. In the present study, hot water extract of A. thracica showed cytotoxic and pro-apoptotic effects on human fibrosarcoma cell line HT1080.
CONCLUSION
Achilea thracica hot and cold water extracts have cytotoxic effect. The HE showed a stronger inhibitory effect on root growth in comparison with the CE. Both extracts exerted mitodepressive effect on A. cepa meristematic cells at concentrations equal to ½ ??50 and EC50 values. Achilea thracica hot water extract exerted a significant dose-dependant negative impact on HT1080 cell viability and showed promising pro-apoptotic activity.
ACKNOWLEDGMENT
This work has been supported by the Bulgarian Ministry of Education and Science, grant no. RD-08-113/20.02.2023.
AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
DATA AVAILABILITY
All data generated and analyzed are included in this research article.
PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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