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Purpose: The phytochemical profile and anticancer potential of three Ajuga sp.
Materials and Methods: The phytochemicals were extracted from the aerial parts of Ajuga sp. The phytochemical profile was also evaluated by principal component analysis in connection with antitumor efficacy of extracts.
Western Blot with regard to inflammatory protein NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit expression in cell lysates was performed.
Enzymatic and non-enzymatic antioxidant capability was assessed by measuring catalase activity and by evaluating the total antioxidant capacity TAC of treated cells.
Results: Ajuga laxmannii ethanol extract showed the highest total phenolic and flavonoid content, while A. The overall cytostatic effect of the investigated plant extracts was exerted through strong inhibitory actions on NF-κB, the key molecule involved in the inflammatory response and via oxidative stress modulatory effects in both murine colon carcinoma and melanoma cell lines.
Conclusion: Ajuga laxmannii showed the most significant antitumor activity and represents an important source of bioactive compounds, possibly an additional form of treatment alongside conventional anticancer drugs. Introduction Medicinal plants have always been an important source for various pharmaceuticals since ancient times. Nowadays the scientific interest for new drugs production from bioactive compounds isolated from natural products is pancreatic cancer can it be cured growing.
Herbal medicines were often used only based on empirical observations since antiquity, without knowing the phytochemicals from the extracts or details of their pharmacological effects Atanasov et al. Although many herbal remedies have a well-known composition and certain biological effects, some of them are still used only based on traditional medicine, and lacking the validation of their safety and efficacy.
The research on unexplored medicinal plants traditionally used in folk medicine could determine the development of novel herbal formulations with significant biological activities. Due to their important pharmacological effects, the natural compounds are effectively used to obtain new phytomedicines.
Ajuga species Lamiaceaewhich are widely distributed in many parts of the world Atay et al. Six Ajuga species are mentioned in the Romanian spontaneous flora, with Ajuga genevensis L. Our previous research showed the antioxidant, antimicrobial, and anti-inflammatory effects of aerial parts extracts Toiu et al.
The monoterpene glycosides content and the essential oil composition have been recently studied on species from Italy, together with the evaluation of antioxidant activity and cytotoxicity by MTT assay Venditti et al. The anticancer activity of natural compounds is attributed to their synergistically acting complex mixture of phytochemicals with chemopreventive and chemotherapeutic potential, which can prove to be far more effective than isolated bioactive molecules de Kok et al.
Accordingly, the unexplored plants used in folk medicine require extensive studies for reliable evidence-based phytotherapy. Although complementary and alternative ethnopharmacological pancreatic cancer can it be cured are mainly focused on counteracting the side effects and collateral symptoms of conventional cancer therapies, in this paper we investigated a potential disjunction change in traditional plant use Leonti and Casu,by assessing the anticancer activity of these indigenous herbs.
Therefore, this study was aimed to perform a comparative phytochemical analysis of A. F10 murine melanoma and C26 colon carcinoma cells. Both cell lines are characterized by increased metastatic potential and are prone to therapeutic alterations of their redox status Rauca et al. In addition, melanoma and colon carcinoma are two of the deadliest cancers in modern society, possibly interlinked by epigenetic mechanisms, as recent reports concluded that colorectal cancer is one of the most common discordant cancers post-melanoma Frank et al.
As previously reported, the highly metastatic B The potential synergistic interaction between bioactive constituents suggests that the whole plant extract may contribute to better therapeutic outcomes compared to the administration of single isolated compounds at an equivalent dose Rasoanaivo et al.
Materials and Methods Chemicals and Reagents High purity chemicals: sodium carbonate, sodium acetate trihydrate, and anhydrous aluminum chloride were acquired from Sigma-Aldrich Germany.
Folin-Ciocâlteu reagent was purchased from Merck Germany. HPLC grade solvents methanol, acetonitrile, ammonium acetate, and silver nitrate were purchased from Sigma-Aldrich. Preparation of Standard Solutions Standard stock solutions of the flavonoids and iridoids were prepared by dissolving 1 mg of each compound in 1 mL methanol and stored at 4°C, protected from daylight.
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They were appropriately diluted with double distilled water before being used as working solutions. Plant Samples and Extraction Procedures The aerial parts of Ajuga species collected in flowering stage in June were obtained and authenticated by one of us A. The dried plant samples were ground to a fine powder before extraction. The aerial parts extracts of A. The liquid chromatograph was equipped with binary gradient pump, degasser, column thermostat and autosampler.
The chromatographic separation was performed on a reversed-phase Zorbax SB-C18 mm × 3.
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The column temperature was set at 48°C. The MS system operated using an electrospray ion source in negative mode. The identification and quantification of polyphenols were made in UV assisted by MS. Quantitative determinations were performed using an external standard method.
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In order to determine the concentration of polyphenols in plant samples, the calibration curves in the range of 0. The compounds were identified by comparison of their retention times and the recorded ESI-MS with those of standards in the same chromatographic conditions Vlase et al.
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The LC was equipped with a binary pump, autosampler, thermostat and detector all Series from Agilent Inc. The system was controlled with Data Analysis software version B For quantitation of the iridoids, stock solutions pancreatic cancer can it be cured the five commercially available standards were prepared in acetonitrile. Cell Proliferation Assay To determine the effect of Ajuga sp.
The range of concentrations for each extract was selected based on previous studies regarding in vitro cytotoxic activity of Ajuga sp. Sadati et al.
To screen for pancreatic cancer can it be cured toxicity, cells were incubated with the same concentrations of the solvent as those used for the preparation of the ethanolic extracts. Cell proliferation was calculated as percentage of untreated cells control value. To measure the effectiveness of the treatments, the IC50 was calculated by GraphPad Prism version 6 for Windows software.
F10 cells were used for total cell lysates preparation as described previously Licarete et al. The homogenates were incubated for 30 min on ice and then centrifuged for 10 min at 15 × g, at 4°C. The supernatants were collected pancreatic cancer can it be cured stored at °C for molecular investigations Rauca et al.
Western Blot Analysis of the Expression Levels of NF-κB-p65 Subunit To assess the effect of the selected ethanolic extracts on the expression of key inflammatory transcription factor NF-κB-p65 subunit in the cell lysates obtained from standard C26 and B F10 cell culture, western blot analysis was performed, as previously described Patras et al.
Electrophoresis was performed at 50 mV, and the electro-transfer of proteins onto a nitrocellulose membrane was conducted at mV for 50 min.
The analysis of the films was performed using Image J freeware for Windows 7 64 bit Licarete et al. The final results were presented as mean ± standard deviation SD of two independent experiments.
Measurement of Oxidative Stress Parameters Malondialdehyde levels in cell lysates were determined by high-performance liquid chromatography HPLC as previously described Licarete et al. The retention time of MDA was about 2. Each sample was determined in duplicate.
The samples were measured in duplicate. Prior to model development the X dataset, represented by the phytochemical composition of each extract, and Y dataset, represented by a binary pancreatic cancer can it be cured matrix encoding class membership were scaled to unit variance. Class membership of observations was assigned in function of plant species.
Model performance was evaluated through the fraction of explained variability by each component R2Xthe total variation of Y explained by the model R2Yand predictive capacity Q2 calculated using full cross-validation.
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Correlations between biological activity and extract type were evaluated using PLS method, through an experimental design approach Modde 11 Pro, Sartorius Stedim, Sweden. The response was represented by the percentage of proliferation inhibition against the control group untreated cells. Model performance was evaluated using R2, Q2, Validity and Reproducibility parameters, while model significance and lack of fit were assessed using F-testing.
Interpretations were done by generating coefficient plots, and the significance of each coefficient factor was tested using ANOVA. Statistical Analysis All phytochemical assays were performed in triplicate, and the results were expressed as the mean ± S.
Analyses were performed using SPSS Data from different experiments were indicated as mean ± SD. The antioxidant and anti-inflammatory effects of the selected Ajuga sp.
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In all cases the ethanol extracts contained higher amounts of each class of bioactive compounds compared to methanol extracts. The content in total iridoids was similar in A. Total phenolic, flavonoid, and iridoid content in A. The analysis of the Ajuga sp. The obtained results allow the characterization of Ajuga sp. The identified compounds: ferulic acid 1isoquercitrin 2rutin 3quercitrin 4luteolin 5and apigenin 6. Polyphenolic profile of A. Iridoid profile of A. Antiproliferative Activity of Ajuga sp.
Extracts on C26 and B F10 cells were expressed as percentage of inhibition compared to the proliferation of the untreated control cells Figures 4A,B and as IC50 values for each extract tested Table 4. The relationship between input variables plant species, extract concentration, cell type — X dataset and cell proliferation inhibition rate Y dataset was assessed by fitting a polynomial equation through PLS method Figure 5.
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The specific types of polyphenols isoquercitrin, rutin and apigenin Table 2 and iridoids harpagoside and 8-O-acetyl-harpagide Table 3 might be involved in strong antitumor activity of the vegetal extracts tested. Effects of Ajuga sp. A 24 h after incubation of B Data are shown as mean ± SD of triplicate measurements. EEAG, A. Ethanol-treated cells were used as toxicity controls. Cytotoxicity of Ajuga sp. Scaled and centered coefficient bar plot for cell proliferation inhibition.
To analyze the effect of factors on cell proliferation inhibition, the coefficients of the polynomial equation were plotted. The expression of NF-κB-p65 in cell lysates after different treatments. F10 cells and C,G C26 cells after different treatments; β-actin was used as loading control. On B Results represent the mean ± SD of two independent measurements. Modulatory Effects of Ajuga sp. Thus, the levels of a general oxidative stress marker — MDA, as well as the catalytic activity of catalase and production of non-enzymatic antioxidant systems were assessed on both cell lines and are shown in Figures 78.
F10 increased the pro-oxidative damage Figures 7B8B in correlation with a proportional increase in the antioxidant capacity of the remaining cancer cells Figures 7F8F of both cell lines. The activity of the antioxidant enzyme catalase on both cell lines was not significantly modified by the IC80 extract concentrations used in this investigation Figures 7D8D.
On C26 cells, the IC50 extract concentrations did not significantly modify any of the parameters of oxidative stress tested Figures 7A,C,E. One way ANOVA test with Bonferroni correction for multiple comparisons was performed to analyze the differences between the effects of the treatments applied on MDA and non-enzymatic antioxidant defense systems levels and on catalase activity.
F10 melanoma cells. E,F Total non-enzymatic antioxidant system levels in the cell lysates obtained from standard culture of B Control, untreated B Multivariate Data Analysis The model built to analyze the phytochemical composition of ethanolic extracts of the three different species consisted of paraziticka ryba predictive components and one orthogonal component.
Both predictive components were found significant judging from their eigenvalues, first predictive component presented an eigenvalue of From the score scatter plot presented under Figure 9a good separation of observations can be seen in function of class membership, reflected also in the total sum of variation in Y explained by the model R2Y- Each observation is represented by a point in multidimensional space, differentially colored according to plant species.
R2X-fraction of variability in X physicochemical descriptors explained by each component; R2Y-total variation of Y class membership explained by the model; Q2-predictive capacity. To identify the phytochemical descriptors responsible for class separation the corresponding loading plot was generated Figure In a pancreatic cancer can it be cured way, the left hand-group of variables represented by rutin, harpagoside, apigenin, isoquercitrin, quercitrin are found in higher concentration in extracts EEAL, respectively in lower concentration in extract EEAG and intermediate concentration in extract EEAC.
The second predictive component captures variability related to ferulic acid and luteolin content, respectively TIC. The relationship between input variables plant species, extract concentration, cell type — X dataset and cell proliferation inhibition rate Y dataset was assessed by fitting a polynomial equation through PLS method.
The developed model explained most of the response variation R The residuals were further decomposed into model error pancreatic cancer can it be cured replicate error and compared.
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According to p value of 0. The model validity parameter 0. To analyze the effect of factors on cell proliferation inhibition, the coefficients of the polynomial pe pleoapele papilomelor were plotted Figure 5. Discussion In the present study, we evaluated the antiproliferative potential of three indigenous Ajuga sp.
As shown in Figures 4A,Bstandardized ethanolic extracts of the three selected species induced different degrees of proliferation inhibition in both cell lines after 24 h.