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In Vitro Antioxidant and Anti-Proliferative Activity of Ranunculaceae Species from Romania

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In Vitro Antioxidant and Anti-Proliferative Activity of Ranunculaceae Species from Romania In vitro antioxidant and anti-proliferative activity of Ranunculaceae species from Romania Cristina Daniela Kelemen, PhD student Department of Horticulture, Faculty of Horticulture, University of Agricultural Sciences and Veterinary Medicine, Manastur Street 3-5, 400372, Cluj-Napoca, Romania; Plants are considered as: rich source of antioxidant compounds (phenolics, anthocyanins, flavonoids); consumption of plant-based antioxidants could be connected with lowered risk of occurrence of several human diseases related to the oxidative stress, including cancer. providers of anti-proliferative compounds (podophyllotoxin, vincristine), which are able to actively inhibit growth of carcinoma cells. The identification of new phytochemical compounds and new plants with antioxidant and anti-proliferative activities remains a priority. Various phytochemical compounds, which shown antioxidant and anti-proliferative activity have been found in Ranunculaceae species: alkaloids glycosides triterpenoid saponins steroids Aconitine Ranunculaceae family is represented by ca 2,500 species in 56 genera distributed worldwide; Nigella sativa in Romania are 23 genera and ca 110 species including rare and endemic plants Local species have been used as: medicinal plants ornamentals locally consumed as food Ranunculus ficaria Antioxidant and anti-proliferative activities were previously reported in many species belonging to the Ranunculaceae family Anemone cathayensis Aconitum heterophyllum Nigella sativa Ranunculus arvensis Antioxidant and anti- In vitro and in vivo antioxidant Antioxidant, anticancer Antioxidant and anti- proliferative activities activity anti-inflammatory, and proliferative activities ( Wang et al., 2012) (Konda, et. al., 2016) antibacterial activities (Bhatti, et al., 2011) (Bourgou, et al., 2012) Antioxidant and anti-proliferative activities remains unknown for many species of the family Aim of the Study: To evaluate the antioxidant and anti-proliferative potentials of 11 Ranunculaceae species used in folk medicine. R. carpaticus R. platanifolius R. repens Aconitum variegatum Aconitum vulparia Anemone transsilvanica Plant Material 11 Ranunculaceae species were collected in summer of 2016 in two locations (Mt. Stramba and Mt. Postavaru) in Romania; Voucher specimens have been stored in the Herbarium collection at USAMV of Cluj- Napoca, Romania. Plants were selected following the ethnobotanical appraisal and medicinal use Table 1: (Ethno)botanical data on Ranunculaceae species tested Species VSN Folk medicine use 1. Aconitum moldavicum Hacq. CLA30049 Analgesic, antitussive 2. Aconitum variegatum L. CLA30048 Analgesic 3. Aconitum vulparia Rchb. CLA30046 Antirheumatic, neuralgia, chronic skin disorders 4. Anemone transsilvanica Fuss. CLA30047 Antibronchitis, anti-hepatitis, antiinfectives 5. Ranunculus acer L. CLA30042 Antirheumatic, antispasmodic, diaphoretic, analgesic 6. Ranunculus bulbosus L. CLA30050 Antispasmodic, dermatosis, rubefacient 7. Ranunculus carpaticus Herbich CLA30044 Analgesic, antirheumatic 8. Ranunculus nemorosus DC. CLA30043 Tonic, antirheumatic 9. Ranunculus platanifolius L. CLA30040 Antirheumatic, antispasmodic, diaphoretic, analgesic 10. Ranunculus polyanthemos L. CLA30051 Gastric and duodenal ulcers 11. Ranunculus repens L. CLA30045 Analgesic, antirheumatic, rubefacient Methodology: Preparation of Ethanolic extracts 5 g of powder of each species were macerated with 150 mL of 80% ethanol for 24 hours at room temperature; The extracts were filtered and the ethanol Ethanolic extracts after maceration was evaporated; Dried residues were dissolved in 100% DMSO* to obtain a stock concentration of 51.2 mg/ml. Rotary vaccum evaporator Dried residues (R-200 Buchi, Switzerlandat). Vacuum filtering apparatus *DMSO- Dimethyl sulfoxide Methodology: Antioxidant assay DPPH* assay described by Sharma 15 μL of extract (512mg/ml) + 1660 μL methanol and Bhat (2009) was used to test the + 25 μL DPPH radical antioxidant activity; 30 min in the dark Sample gets reduced Color change from deep violet to light yellow Absorbance 520 nm The synthetic antioxidant Trolox was used as a positive control. *1,1-diphenyl-2-picrylhydrazyl (DPPH) assay of Ranunculaceae samples Methodology: Anti- proliferative assay MTT* cytotoxicity method- Caco-2, HT-29 and FHs-74 Int cell lines Mosmann (1983) were incubated for 24 h in EMEM medium Cell lines were treated with serial extracts metabolization dilutions (16–512μg/mL) for 72 h . Spent medium was replaced by fresh MTT- yellow Formazan- blue EMEM containing MTT (1mg/mL) and incubated for an 2 h Formazan product was dissolved in DMSO Absorbance 555 nm *MTT-(3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide) Results: Antioxidant activity. of Ranunculaceae species Table 2. IC50 values of ethanolic extracts of Ranunculaceae species The means were expressed as Antioxidant assays/ DPPH fre radical scavenging assay half maximal inhibitory Species name IC (μg/mL) ± SD concentration (µg/mL IC ) 50 50 A. moldavicum 123,09 ± 26,0 Aconitum species exhibits A. variegatum 137,02 ± 6,5 antioxidant activity namely: A. vulparia 114,04 ± 14,9 A. transsilvanica 179,57± 12,9 A. vulparia (IC50:114 μg/mL), R. acer 161,70± 24,3 A. moldavicum (IC50:123μg/mL) R. bulbosus 166,87 ± 10,1 A. variegatum (IC50:137 μg/mL). R. carpaticus 207,90 ± 2,02 R. nemorosus 240,53 ± 12,7 R. platanifolius >268.79 R. polyanthemos 157,77 ± 17,2 R. repens 190,06 ± 17,9 Trolox 14,68 ± 3,4 *IC50 half maximal inhibitory concentration; *The values are mean ± SD (n = 3) Results: Anti-proliferative activity of . Ranunculaceae species Tab 3. IC50 values for anti-proliferative activity of Ranunculaceae extracts Caco-2 HT-29 Species name FHs-74 Int IC50(µg/mL) IC50(µg/mL) A. moldavicum 160.66 ± 7.50 77.54 ± 10.45 291.25 ± 7.21 A. variegatum 144.29 ± 7.54 330.66 ± 17.89 >512 A. vulparia 249.87 ± 0.80 184.54 ± 2.05 >512 46.88 ± 5.94 86.47 ± 4.46 154.84 ± 17.16 A. transsilvanica 65.77 ± 2.63 70.24 ± 9.37 259.64 ± 19.10 R. acer >512 205.25 ± 15.44 450.29 ± 12.08 R. bulbosus 152.27 ± 6.29 221.92 ± 5.60 285.14 ± 6.36 R. carpaticus 277.60 ± 9.58 387.68 ± 0.51 318.93 ± 19.8 R. nemorosus 83.34 ± 4.62 67.39 ± 1.81 152.97 ± 4.63 R. platanifolius 242.45 ± 6.81 253.15 ± 0.42 >512 R. 318.23 ± 5.81 230.66 ± 9.27 >512 polyanthemos R. repens 227.61 ± 5.98 295.19 ± 18.04 >512 *IC50 half maximal inhibitory concentration; *The values are mean ± SD (n = 3) Aconium species exhibits antioxidant activity with IC50 values 114-137 µg/mL. A. moldavicum, A. transsilvanica and R. nemorosus possessed significant anti-proliferative activity against Caco-2 and HT-29 cells with IC50 range from 144.2 to 387.6 µg/mL . These species can be considered as prospective materials for the further development of novel plant- based antioxidant and/or anti-proliferative agents. A. moldavicum Detailed analysis of their chemical composition and in vivo antioxidant/anti-proliferative activity should be carried out in order to verify their possible practical use. REFERENCES 1. Heywood H., Brummitt R. K., Culham A., Seberg O.V., 2007. Flowering Plant Families of the World. Edited by R.V.H. Heywood. Firefly Bo. Ontario. 2. Tămaș M., (2005.) Botanică Farmaceutică: Sistematica-Cormobionta, 3th edn. Editura Medicală Universitară Publishing House, Cluj Napoca, 40-44. 3. Bhatti, M. Z., Ali, A., Saeed, A., Saeed, A. (2011). Antimicrobial , antitumor and brine shrimp lethality assay of Ranunculus arvensis L . extracts, Pak. J. Pharm. Sci., 28 (3), 945–949. 4. Bourgou, S., Pichette, A., Marzouk, B., Legault, J. (2012). Antioxidant, anti-inflammatory, anticancer and antibacterial activities of extracts from nigella sativa (black cumin) plant parts. Journal of Food Biochemistry, 36, 539–546. 5. Konda, V. G. R., Eerike, M., Raghuraman, L. P., Rajamanickam, M. K. (2016). Antioxidant and nephroprotective activities of aconitum heterophyllum root in glycerol induced acute renal failure in rats. Journal of Clinical and Diagnostic Research, 10(3), 1–5. 6. Wang, J. li, Liu, K., Gong, W. zhen, Wang, Q., Xu, D. ting, Liu, M. Fei, Song, Y. Fei. (2012). Anticancer, antioxidant, and antimicrobial activities of anemone (Anemone cathayensis). Food Science and Biotechnology, 21(2), 551–557. 7. Wang, X.-Y., Gao, H., Xie, X.-J., Jurhiin, J., Zhang, M.-Z.-H., Zhou, Y.-P., Tang, H.-F. (2018). Triterpenoid Saponins from Anemone rivularis var. Flore-Minore and Their Anti-Proliferative Activity on HSC-T6 Cells. Molecules, 23(2), 491. 8. 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