In vitro antioxidant and anti-proliferative activity of Ranunculaceae species from Romania Cristina Daniela Kelemen, PhD student

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Cristina Daniela Kelemen, PhD student Department

1 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, , Cluj-Napoca, Romania;

Plants are considered as: rich source of antioxidant compounds

antioxidants could be connected with lowered risk of occurrence of

vincristine), which are able to actively inhibit growth of

2 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

triterpenoid saponins steroids Aconitine Ranunculaceae family is represented by

and ca 110 species including rare and endemic plants Nigella sativa Local

3 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; in Romania are 23 genera and ca 110 species including rare and endemic plants Nigella sativa 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

, 2016) Nigella sativa Antioxidant, anticancer anti-inflammatory, and antibacterial activities

4 Antioxidant and anti-proliferative activities were previously reported in many species belonging to the Ranunculaceae family Anemone cathayensis Antioxidant and antiproliferative activities ( Wang et al., 2012) Aconitum heterophyllum In vitro and in vivo antioxidant activity (Konda, et. al., 2016) Nigella sativa Antioxidant, anticancer anti-inflammatory, and antibacterial activities (Bourgou, et al., 2012) Ranunculus arvensis Antioxidant and antiproliferative activities (Bhatti, et al., 2011) Antioxidant and anti-proliferative activities remains unknown for many species of the family

5 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

6 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

7 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; Ethanolic extracts after maceration The extracts were filtered and the ethanol was evaporated; Rotary vaccum evaporator (R-200 Buchi, Switzerlandat). Dried residues Dried residues were dissolved in 100% DMSO* to obtain a stock concentration of 51.2 mg/ml. Vacuum filtering apparatus *DMSO- Dimethyl sulfoxide

(512mg/ml) + 1660 μl methanol + 25 μl DPPH radical 30 min in the dark Sample gets reduced Color change

8 *1,1-diphenyl-2-picrylhydrazyl (DPPH) assay of Ranunculaceae samples Methodology: Antioxidant assay DPPH* assay described by Sharma and Bhat (2009) was used to test the antioxidant activity; 15 μl of extract (512mg/ml) μl methanol + 25 μl DPPH radical 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.

9 Methodology: Antiproliferative assay MTT* cytotoxicity method- Mosmann (1983) Caco-2, HT-29 and FHs-74 Int cell lines were incubated for 24 h in EMEM medium metabolization Cell lines were treated with serial extracts dilutions (16 512μg/mL) for 72 h. MTT- yellow Formazan- blue Spent medium was replaced by fresh 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)

10 . Results: Antioxidant activity of Ranunculaceae species The means were expressed as half maximal inhibitory concentration (µg/ml IC 50 ) Aconitum species exhibits antioxidant activity namely: A. vulparia (IC 50 :114 μg/ml), A. moldavicum (IC 50 :123μg/mL) A. variegatum (IC 50 :137 μg/ml). Table 2. IC 50 values of ethanolic extracts of Ranunculaceae species Antioxidant assays/ DPPH fre radical scavenging assay Species name IC 50 (μg/ml) ± SD A. moldavicum 123,09 ± 26,0 A. variegatum 137,02 ± 6,5 A. vulparia 114,04 ± 14,9 A. transsilvanica 179,57± 12,9 R. acer 161,70± 24,3 R. bulbosus 166,87 ± 10,1 R. carpaticus 207,90 ± 2,02 R. nemorosus 240,53 ± 12,7 R. platanifolius > R. polyanthemos 157,77 ± 17,2 R. repens 190,06 ± 17,9 Trolox 14,68 ± 3,4 *IC 50 half maximal inhibitory concentration; *The values are mean ± SD (n = 3)

11 Results: Anti-proliferative activity of. Ranunculaceae species Tab 3. IC 50 values for anti-proliferative activity of Ranunculaceae extracts Species name Caco-2 HT-29 IC 50 (µg/ml) IC 50 (µg/ml) FHs-74 Int A. moldavicum ± ± ± 7.21 A. variegatum ± ± >512 A. vulparia ± ± 2.05 >512 A. transsilvanica ± ± ± ± ± ± R. acer > ± ± R. bulbosus ± ± ± 6.36 R. carpaticus ± ± ± 19.8 R. nemorosus ± ± ± 4.63 R. platanifolius ± ± 0.42 >512 R. polyanthemos ± ± 9.27 >512 R. repens ± ± >512 *IC 50 half maximal inhibitory concentration; *The values are mean ± SD (n = 3)

Aconium species exhibits antioxidant activity with IC 50 values 114-137

nemorosus possessed significant anti-proliferative activity against

These species can be considered as prospective materials for the further

12 Aconium species exhibits antioxidant activity with IC 50 values µg/ml. A. moldavicum, A. transsilvanica and R. nemorosus possessed significant anti-proliferative activity against Caco-2 and HT-29 cells with IC 50 range from to µg/ml. These species can be considered as prospective materials for the further development of novel plantbased antioxidant and/or anti-proliferative agents. 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. A. moldavicum

13 REFERENCES 1. Heywood H., Brummitt R. K., Culham A., Seberg O.V., 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, 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), 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, 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), 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), 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), Nuntanakorn P., Jiang B., Yang H., Cervantes M., Kronenberg F., Kennelly E.L., (2007) Analysis of polyphenolic compounds and radical scavenging activity of four American Actaea species. Phytochemical Analysis, 18(3), pp Mostafa, M., Afolayan, A.J., (2013) Antimicrobial activity of Clematis brachiata thunb leaf extracts. International Journal of Pharmaceutical Sciences and Research, 4(1), pp Srivastava N., Sharma V., Saraf K., Dobriyal A.K., Kamal B., Jadon V.S., (2011) In vitro antimicrobial activity of aerial parts extracts of Aconitum heterophyllum Wall. ex Royle Nidhi. Indian Journal of Natural Products and Resources, 2(4), pp

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DEMETRIOS KOURETAS PROFESSOR DEPARTMENT OF BIOCHEMISTRY & BIOTECHNOLOGY UNIVERSITY OF THESSALY, GREECE

DEMETRIOS KOURETAS PROFESSOR DEPARTMENT OF BIOCHEMISTRY & BIOTECHNOLOGY UNIVERSITY OF THESSALY, GREECE Entrepreneurial Discovery Focus Group on wine for Eastern Macedonia and Thrace Drama, Greece Vitis