Phytochemical screening of Pulsatilla species and investigation of their biological activities

Grażyna Łaska, Aneta Sienkiewicz, Marcin Stocki, Jordan K. Zjawiony, Vimal Sharma, Andrzej Bajguz, Alicja Piotrowska-Niczyporuk, Melissa Jacob, Shabana Khan

Abstract


The present study aimed to identify biologically active secondary metabolites from the rare plant species, Pulsatilla patens subsp. patens and the cultivated P. vulgaris subsp. vulgaris. Chromatographic fractionation of the ethanolic extract of the roots of P. patens subsp. patens resulted in the isolation of two oleanane-type glycosides identified as hederagenin 3-O-β-d-glucopyranoside (2.7 mg) and hederagenin 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucopyranoside (3.3 mg, patensin). HPLC analysis of the methanolic extract of the crude root of P. patens subsp. patens and P. vulgaris subsp. vulgaris revealed the presence of Pulsatilla saponin D (hederagenin 3-O-α-l-rhamnopyranosyl(1→2)-[β-d-glucopyranosyl(1→4)]-α-l-arabinopyranoside). Chromatographic analysis using GC-MS of the silylated methanolic extracts from the leaves and roots of these species identified the presence of carboxylic acids, such as benzoic, caffeic, malic, and succinic acids. The extracts from Pulsatilla species were tested for their antifungal, antimicrobial, and antimalarial activities, and cytotoxicity to mammalian cell lines. Both P. patens subsp. patens and P. vulgaris subsp. vulgaris were active against the fungus Candida glabrata with the half-maximal inhibitory concentration (IC50) values of 9.37 µg/mL and 11 µg/mL, respectively. The IC50 values for cytotoxicity evaluation were in the range of 32–38 μg/mL for P. patens subsp. patens and 35–57 μg/mL for P. vulgaris subsp. vulgaris for each cell line, indicating general cytotoxic activity throughout the panel of evaluated cancer and noncancer cells.

Keywords


pasque flower subspecies; oleanane-type triterpenoid glycosides; carboxylic acids; antifungal and antimicrobial activities; cytotoxicity; medicinal applications

Full Text:

PDF

References


Kim Y, Bang SC, Lee JH, Ahn BZ. Pulsatilla saponin D: the antitumor principle from Pulsatilla koreana. Arch Pharm Res. 2004;27(9):915–918. https://doi.org/10.1007/BF02975843

Bang SC, Kim Y, Lee JH, Ahn BZ. Triterpenoid saponins from the roots of Pulsatilla koreana. J Nat Prod. 2005;68(2):268–272. https://doi.org/10.1021/np049813h

Seo KH, Jung JW, Thi NN, Lee YH, Baek NI. Flavonoid glycosides from the flowers of Pulsatilla koreana Nakai. Natural Product Sciences. 2016;22(1):41–45. https://doi.org/10.20307/nps.2016.22.1.41

Shumova GS, Savelieva EV, Vladymyrova IN, Tishakova TS. Phenolic compound composition of herb of Pulsatilla pratensis (L.) Mill. World Science. 2017;12(28):35–38.

Shu Z, Chen Z, Liu YL, Zhu WF, Feng YL, Xu QM, et al. A new oleanane-type triterpenoidal saponin from Pulsatilla chinensis. Nat Prod Res. 2013;27(23):2196–2201. https://doi.org/10.1080/14786419.2013.814052

Yang H, Cho YW, Kim SH, Kim YC, Sung SH. Triterpenoidal saponins of Pulsatilla koreana roots. Phytochemistry. 2010;71(16):1892–1899. https://doi.org/10.1016/j.phytochem.2010.07.013

Fan W, Liu J, Gong Y, Ma J, Zhou N, Xu Y. A new triterpenoid saponin from Pulsatilla cernua. Natural Product Sciences. 2013;19(2):150–154.

Sun H, Wang Y, Zhang XQ, Zhao SX, Ye WC. Chemical constituents of Pulsatilla dahurica. Chem Nat Compd. 2009;45(5):764–765. https://doi.org/10.1007/s10600-009-9454-3

Xu HJ, Shi XW, Ji X, Du YF, Zhu H, Zhang LT. A rapid method for simultaneous determination of triterpenoid saponins in Pulsatilla turczaninovii using microwave-assisted extraction and high performance liquid chromatography-tandem mass spectrometry. Food Chem. 2012;135(1):251–258. https://doi.org/10.1016/j.foodchem.2012.04.081

Samadder A, Das J, Das S, Khuda-Bukhsh AR. Dihydroxy-isosteviol-methyl-ester, an active biological component of Pulsatilla nigricans, reduces arsenic induced cellular dysfunction in testis of male mice. Environ Toxicol Pharmacol. 2012;34(3):743–752. https://doi.org/10.1016/j.etap.2012.09.013

Rolski S, Przyborowski L. Dissertationes Pharmaceuticae. 1961;13:349–355.

Ye WC, Ji NN, Zhao SX, Che CT. A new cytotoxic saponin from Pulsatilla patens var. multifida. Pharm Biol. 2001;39(1):7–10. https://doi.org/10.1076/phbi.39.1.7.5951

Danova K, Bertoli A, Pistelli L, Dimitrow D, Pistelli L. In vitro culture of Balkan endemic and rare Pulsatilla species for conservational purposes and secondary metabolites production. Bot Serb. 2009;33(2):157–162.

Xu QM, Shu Z, He WJ, Chen LY, Yang SL, Yang G, et al. Antitumor activity of Pulsatilla chinensis (Bunge) Regel saponins in human liver tumor 7402 cells in vitro and in vivo. Phytomedicine. 2012;19(3–4):293–300. https://doi.org/10.1016/j.phymed.2011.08.066

Yoo HH, Lee SK, Lim SY, Kim Y, Kang MJ, Kim EJ, et al. LC-MS/MS method for determination of hederacolchiside E, a neuroactive saponin from Pulsatilla koreana extract in rat plasma for pharmacokinetic study. J Pharm Biomed Anal. 2008;48(5):1425–1429. https://doi.org/10.1016/j.jpba.2008.09.012

Liu JY, Guan YL, Zou LB, Gong YX, Hua HM, Xu YN, et al. Saponins with neuroprotective effects from the roots of Pulsatilla cernua. Molecules. 2012;17:5520–5531. https://doi.org/10.3390/molecules17055520

Dai L, Wang H, Chen Y. The immune-enhancing effect of PcG-A-a glycoprotein isolated from dried root of Pulsatilla chinensis (Bunge) Regel. Chinese Journal of Biochemical Pharmaceutics. 2000;21:230–231.

Seo JS, Yun JH, Baek IS, Leem YH, Kang HW, Cho HK, et al. Oriental medicine Jangwonhwan reduces Aβ(1–42) level and β-amyloid deposition in the brain of Tg-APPswe/PS1dE9 mouse model of Alzheimer disease. J Ethnopharmacol. 2010;128:206–212. https://doi.org/10.1016/j.jep.2010.01.014

Li HB, Wong CC, Cheng KW, Chen F. Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. LWT – Food Science and Technology. 2008;41:385–390. https://doi.org/10.1016/j.lwt.2007.03.011

Lee HS, Beon MS, Kim MK. Selective growth inhibitor toward human intestinal bacteria derived from Pulsatilla cernua root. J Agric Food Chem. 2001;49:4656–4661. https://doi.org/10.1021/jf010609z

Xu K, Shu Z, Xu QM, Liu YL, Li XR, Wang YL, et al. Cytotoxic activity of Pulsatilla chinensis saponins and their structure-activity relationship. J Asian Nat Prod Res. 2013;15(6):680–686. https://doi.org/10.1080/10286020.2013.790901

Nelson LS, Shih RD, Balick MJ. Handbook of poisonous and injurious plant. New York, NY: The New York Botanical Garden; 2007.

Wink M, van Wyk BE. Mind-altering and poisonous plants of the world. A scientifically accurate guide to 1,200 toxic and intoxicating plants. Portland, OR: Timber Press; 2008.

Vance JC. Toxic plants of Minnesota: skin toxicity of the Prairie Crocus (Anemone patens L.). Minn Med. 1982;65:149–151.

Turner NJ. Counter-irritant and other medicinal uses of plants in Ranunculaceae by native peoples in British Columbia and neighbouring areas. J Ethnopharmacol. 1984;11:181–200. https://doi.org/10.1016/0378-8741(84)90038-2

Encyclopedia of Chinese materia medica. Shanghai: Shanghai Science and Technology Press; 1977.

Szczecińska M, Sramko G, Wołosz K, Sawicki J. Genetic diversity and population structure of the rare and endangered plant species Pulsatilla patens (L.) Mill. in east Central Europe. PLoS One. 2016;11(3):e0151730. https://doi.org/10.1371/journal.pone.0151730

Piqueray J, Saad L, Bizoux JP, Mahy G. Why some species cannot colonise restored habitats? The effects of seed and microsite availability. J Nat Conserv. 2013;21:189–197. https://doi.org/10.1016/j.jnc.2012.12.005

Kricsfalusy V. Variations in the life cycle of Anemone patens L. (Ranunculaceae) in wild populations of Canada. Plants (Basel). 2016;5(3):E29. https://doi.org/10.3390/plants5030029

Meusel H, Jäger E, Weinert E. Vergleichende Chorologie der Zentraleuropäischen Flora. 1. Karten. Jena: G. Fischer Verl.; 1965.

Den virtuella floran – Naturhistoriska riksmuseet [Internet]. 1996 [cited 2017 Aug 1]. Available from: http://linnaeus.nrm.se/flora/di

Holub J, Procházka F. Red list of vascular plants of the Czech Republic – 2000. Preslia. 2000;72:187–230.

Röder D, Kiehl K. Population structure and population dynamic of Pulsatilla patens (L.) Mill. in relation to vegetation characteristics. Flora. 2006;201:499–507. https://doi.org/10.1016/j.flora.2005.11.001

Gärdenfors U. Population viability analysis in the classification of threatened species: problems and potentials. Ecol Bull. 2000;48:181–190.

Носков [Noskov] ГА [GA], editor. Красная книга природы Ленинградской области. Растения и грибы [Krasnaja kniga prirody Leningradskoj oblasti. Rastenija i griby]. Vol. 2. Санкт-Петербург [Sankt-Peterbúrg]: Мир [Mir]; 2000.

European Nature Information System. Species factsheet for Pulsatilla patens. 2005; Available from: https://eunis.eea.europa.eu/

Průša D, Eliáš MLP, Dítě D, Čačko L, Krása P, Podešva Z, et al. Chránené rastliny Českej a Slovenskej republiky. Brno: Computer Press; 2005.

Rassi P, Alanen A, Kanerva T, Mannerkoski I, editors. The 2000 red list of Finnish species. Helsinki: The II Committee for the Monitoring of Threatened Species in Finland, the Ministry of the Environment; 2001.

Rozporządzenie Ministra Środowiska z dnia 9 października 2014 r. w sprawie ochrony gatunkowej roślin. Journal of Laws of the Republic of Poland (Dziennik Ustaw), 2014 Oct 9, Item 1409.

Zając A, Zając M, editors. Distribution atlas of vascular plants in Poland. Cracow: Laboratory of Computer Chorology, Institute of Botany, Jagiellonian University; 2001.

IUCN. The IUCN Red List of Threatened Species. Version 2014.1 [Internet]. 2014 [cited 2014 Jun 12]. Available from: http://www.iucnredlist.org/

Witkowski ZJ, Król W, Solarz W, editors. Carpathian list of endangered species. Vienna: WWF and Institute of Nature Conservation, Polish Academy of Sciences; 2003.

Gärdenfors U. The 2010 red list of Swedish species. Uppsala: ArtDatabanken, SLU; 2010.

Cheffings C, Farrell L, editors. The vascular plant red data list for Great Britain. Peterborough: Joint Nature Conservation Committee; 2005.

Ludwig G, Schnittler M. Red list of threatened plants in Germany. Bonn: Bundesamt für Naturschutz; 1996.

Moser D, Gygax A, Bäumler B, Wyler N, Palese R. Red list of the threatened ferns and flowering plants of Switzerland. Bern: Bundesamt für Umwelt, Wald und Landschaft, Zentrum des Datenverbundnetzes der Schweizer Flora; 2002.

Niklfeld H, Schratt-Ehrendorfer L, editors. Red list of threatened plants of Austria. Vienna: Grüne Reihe, Bundesministerium für Umwelt, Jugend und Familie; 1999.

Kaźmierczakowa R, Zarzycki K, Mirek Z. Polish red data book of plants. Cracow: W. Szafer Institute of Botany, Polish Academy of Sciences; 2016.

Mirek Z, Piękoś-Mirkowa H, Zając A, Zając M. Flowering plants and pteridophytes of Poland. A checklist. Cracow: W. Szafer Institute of Botany, Polish Academy of Sciences; 2002.

Li H, Li H, Hao N, Xu Y, Piao Z. Study on HPLC fingerprint characteristics and chemotaxonomy of Pulsatilla medicinal plants. China Journal of Chinese Materia Medica. 2011;36(11):1478–1482.

National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard. Wayne, PA: National Committee for Clinical Laboratory Standards; 2002. [NCCLS; vol 22(15)].

National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard. Wayne, PA: National Committee for Clinical Laboratory Standards; 2002. [NCCLS; vol 22(16)].

National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests: approved standard. Wayne, PA: National Committee for Clinical Laboratory Standards; 2003. [NCCLS; vol 23(18)].

National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests: approved standard. Wayne, PA: National Committee for Clinical Laboratory Standards; 2006. [NCCLS; vol 26(2)].

Franzblau SG, Witzig RS, Mclaughlin JC, Torres P, Madico G, Hernandez A, et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol. 1998;36(2):362–366.

Makler MT, Hinrichs DJ. Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am J Trop Med Hyg. 1993;48:205. https://doi.org/10.4269/ajtmh.1993.48.205

Borenfreund E, Babich H, Martin-Alguacil N. Rapid chemosensitivity assay with human normal and tumor cells in vitro. In Vitro Cell Dev Biol. 1990;26:1030. https://doi.org/10.1007/BF02624436

Fidel PL, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev. 1999;12(1):80–96. https://doi.org/10.1128/CMR.12.1.80

Kim J, Sudbery P. Candida albicans, a major human fungal pathogen. J Microbiol. 2011;49(2):171–177. https://doi.org/10.1007/s12275-011-1064-7

Latgé JP. Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev. 1999;12(2):310–350. https://doi.org/10.1128/9781555815523

Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142–201. https://doi.org/10.1128/CMR.11.1.142

Streeter K, Katouli M. Pseudomonas aeruginosa: a review of their pathogenesis and prevalence in clinical settings and the environment. Infect Epidemiol Med. 2016;2(1):25–32. https://doi.org/10.18869/modares.iem.2.1.25

Ye WC, Ou BX, Ji NN, Zhao SX, Ye T, Mc Kervey MA, et al. Patensin, a saponin from Pulsatilla patens var. multifida. Phytochemistry. 1995;39(4):937–939. https://doi.org/10.1016/0031-9422(95)00021-X

Ye W, Pan G, Zhang Q, Che CT, Wu H, Zhao S. Five new triterpene saponins from Pulsatilla patens var. multifida. J Nat Prod. 1999;62(2):233–237. https://doi.org/10.1021/np9802668

Ye WC, Zhang QW, Hsiao WLW, Zhao SX, Che CT. Novel cytotoxic lupane-type glycosides from Pulsatilla chinensis. Planta Med. 2002;68:183–186. https://doi.org/10.1055/s-2002-20254

Chen Z, Guan Y, Zhou L, Xu Y, Yang M, Liu H. Preparation and characterization of colon-targeted particles of Pulsatilla chinensis saponins. Nat Prod Commun. 2015;10(2):237–238.

Li LD, Li WC, Liu CW, Shi WJ, Gong PT, Li JH, et al. Giardia intestinalis: effects of Pulsatilla chinensis extracts on trophozoites. Parasitol Res. 2012;111(5):1929–1935. https://doi.org/10.1007/s00436-012-3035-2

Li WC, Shi WJ, Gu YF, Chen HL, Chen W, Zhang Y. Antibacterial effect of different extract of Pulsatilla chinensis (Bunge) Regel in vitro. Journal of Traditional Chinese Veterinary Medicine. 2011;2:38–40.

Ma C. Treatment methods of traditional Chinese medicines against intestinal protozoan infections. In: Mehlhorn H, Zhongdao W, Ye B, editors. Treatment of human parasitosis in traditional Chinese medicine. Berlin: Springer; 2014. p. 11–21. (Parasitology Research Monographs; vol 6). https://doi.org/10.1007/978-3-642-39824-7_2

Bi YL, Wang B, Huang BH, Zhang WT, Zhang YH. Antifungal activity of botanical extracts against Botrytis cinerea and Alternaria solani. Agricultural Science and Technology. 2011;12(6):862–864.

Chen YQ, Xu QM, Liu YL, Li XR, Yang SL, Zhuge HX. Laboratory evaluation of the molluscicidal activity of Pulsatilla chinensis (Bunge) Regel saponins against the snail Oncomelania hupensis. Biomed Environ Sci. 2012;25(2):224–229. https://doi.org/10.3967/0895-3988.2012.02.015

Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China. Beijing: People’s Medical Publishing House; 2005.

Kim Y, Kim SB, You YJ, Ahn BZ. Deoxypodophyllotoxin; the cytotoxic and antiangiogenic komponent from Pulsatilla koreana. Planta Med. 2002;68:268–271. https://doi.org/10.1055/s-2002-23140

Cheon SA, Choi BK, Jeong CS, Li DW, Lee EB. The anti-inflammatory and analgesic actions of the fractions from Pulsatilla koreana root extract. Korean Journal of Clinical Pharmacy. 2000;31(2):174–184.

Li W, Sun YN, Yan XY, Yang SY, Lee SJ, Byun HJ, et al. Isolation of nematicidal triterpenoid saponins from Pulsatilla koreana root and their activities against Meloidogyne incognita. Molecules. 2013;18:5306–5316. https://doi.org/10.3390/molecules18055306

Chung SW, Chung CH, Lim SB, Kim JK, So EH. The antimicrobial effect of Pulsatilla koreana extracts to oral micro-organism. Journal of the Korean Academy of Periodontology. 2000;30(3):661. https://doi.org/10.5051/jkape.2000.30.3.661

Ling Y, Lin Z, Zha W, Lian T, You S. Rapid detection and characterisation of triterpene saponins from the root of Pulsatilla chinensis (Bunge) Regel by HPLC-ESI-QTOF-MS/MS. Phytochem Anal. 2016;27(3–4):174–183. https://doi.org/10.1002/pca.2613

Lee KY, Cho YW, Park J, Lee DY, Kim SH, Kim YC, et al. Quality control of Pulsatilla koreana based on the simultaneous determination of triterpenoidal saponins by HPLC-ELSD and principal component analysis. Phytochem Anal. 2010;21:314–321. https://doi.org/10.1002/pca.1201

Zhang Y, Bao J, Wang K, Jia X, Zhang C, Huang B, et al. Pulsatilla saponin D inhibits autophagic flux and synergistically enhances the anticancer activity of chemotherapeutic agents against HeLa cells. Am J Chin Med. 2015;43(8):1657–1670. https://doi.org/10.1142/S0192415X15500949

Zhang QW, Ye WC, Che CT, Zhao SX. Triterpene saponins from Pulsatilla cernua. Acta Pharmaceutica Sinica. 2000;35(10):756–759.

Jin ZL, Gao N, Zhou D, Chi MG, Yang XM, Xu JP. The extracts of Fructus Akebiae, a preparation containing 90% of the active ingredient hederagenin: serotonin, norepinephrine and dopamine reuptake inhibitor. Pharmacol Biochem Behav. 2012;100(3):431–439. https://doi.org/10.1016/j.pbb.2011.10.001

Xu HJ, Shi XW, Ji X, Du YF, Zhu H, Zhang LT. Qualitative and quantitative determination of nine main active constituents in Pulsatilla cernua by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. J Sep Sci. 2011;34:308–316. https://doi.org/10.1002/jssc.201000660

Goyal S, Kumar S. Anti-anxiety activity studies of various extracts of Pulsatilla nigricans Stoerck. International Journal of Pharmaceutical Sciences and Drug Research. 2010;2(4):291–293.

Nikolova M, Asenov A. Surface flavonoid aglycones in newly studied plant species. Nat Prod Res. 2006;20:103–106. https://doi.org/10.1080/14786410500046463

Arcus M, Lilios G, Schröder V, Taralunga GH, Stoicescu RM, Dumitru IM. Natural compounds with benefits regarding the cardiovascular diseases. Journal of Environmental Protection and Ecology. 2015;16(1):333–339.

Hegnauer R. Chemotaxonomie der Pflanzen. Stuttgart: Birkhäuser Verlag Basel; 1969.




DOI: https://doi.org/10.5586/asbp.3613

Journal ISSN:
  • 2083-9480 (online)
  • 0001-6977 (print; ceased since 2016)
This is an Open Access journal, which distributes its content under the terms of the Creative Commons Attribution License, which permits redistribution, commercial and non-commercial, provided that the content is properly cited.
The journal is a member of the Committee on Publication Ethics (COPE) and aims to follow the COPE’s principles.
The journal publisher is a member of the Open Access Scholarly Publishers Association.
The journal content is indexed in Similarity Check, the Crossref initiative to prevent scholarly and professional plagiarism.
Publisher
Polish Botanical Society