Secondary metabolites of in vitro cultures of selected Polish rare and endangered plants

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1 B. Thiem, J. Budzianowski, M. Wesołowska, L. Ratajczak, M. Morozowska, L. Skrzypczak 158 Secondary metabolites of in vitro cultures of selected Polish rare and endangered plants Barbara Thiem 1*, JAROMIR Budzianowski 1, MARIA Wesołowska 1, LIDIA Ratajczak 2, MARIA Morozowska 3, LUTOSŁAWA Skrzypczak 1 1 Department of Pharmaceutical Botany and Plant Biotechnology Poznań University of Medical Sciences Św. Marii Magdaleny Poznań, Poland 2 Department of Medicinal and Cosmetic Natural Products Poznań University of Medical Sciences Mazowiecka Poznań, Poland 3 Department of Botany Poznań University of Life Sciences Wojska Polskiego 71C Poznań, Poland *corresponding author: bthiem@ump.edu.pl S u m m a r y A short review on phytochemical analysis of in vitro cultures of selected Polish rare and endangered plants is presented. Analysed secondary metabolites, present in investigated species, are responsible for raw materials medical activity. Phenolic compounds (flavonoids and phenolic acids), secoiridoids, saponins and fatty acids were investigated in micropropagated plants, shoot cultures or callus tissue of eleven plant species from the genera: Gentiana L., Oenothera L., Rubus L., Solidago L., Pinguicula L., Primula L., Eryngium L. The studied species represented medicinal plants or plants used in folk medicine in which the secondary metabolites are usually responsible for their therapeutic properties. The investigations showed that those plants contain pharmaceutically promising compounds. Therefore, plant biotechnology can be used for rapid propagation of rare or endangered plants and the regenerated plants may be used as a source of those rare medicinal plants. Key words: in vitro cultures, micropropagation, secondary metabolites, phytochemical analyses

2 Secondary metabolites of in vitro cultures of selected Polish rare and endangered plants 159 Introduction In order to perform phytochemical studies and characterize the bioactive compounds of some rare plants from Polish flora a constant source of those plants is needed. Furthermore, the natural habitats of these species are constantly shrinking. The accelerating loss of plant species, as a result of destruction of their natural habitat, has yielded a revival of interest in the in vitro propagation of rare and endangered plants [1, 2, 3]. In Poland the technology of in vitro micropropagation and storage is described only for a small number of endangered or threatened plant species [4]. There are two basic strategies of preserving plant genomic reserve: conservation in situ and ex situ. One of the ex situ methods is micropropagation and storage of plants in in vitro cultures. Given that clonal propagation also preserves the genetic stability of the progeny, so in vitro collections are important ex situ germplasm reservoirs [5, 6]. The necessity of cultivation of these species in vitro originates from the need for a constant source of the plant material for phytochemical investigations. In consequence, cultivated plants can serve as an ecologically friendly alternative for phytochemical purposes. Another reason for the necessity of a rapid method of in vitro micropropagation is the slow growth and propagation of that selected plants in conventional methods in the field. The aim of the presented paper is a short review on in vitro cultures and phytochemical analyses of selected Polish rare and endangered plants, i.e. eleven species from the genera: Gentiana L., Oenothera L., Rubus L., Solidago L., Pinguicula L., Primula L. and Eryngium L. Our detailed studies were targeted for the establishment of in vitro techniques for the cultivation of selected plants, especially the development of a micropropagation system and a functioning weaning process for the transfer of in vitro-derived plants to in vivo conditions. The investigation of the presence of secondary metabolites was performed not only for the in vitro propagated plants, but also for cell cultures and callus tissue in vitro cultures to determine their ability to synthesize those metabolites (tab. 1). The secondary metabolites under investigations are usually responsible for the pharmacological properties of the studied medicinal plants. In vitro cultures and conservation of plant germplasm Protocols for micropropagation and callus tissue establishment of eleven selected rare and endangered species were described earlier for Gentiana cruciata L., Gentiana punctata L. [7], Oenothera ammophila Focke, O. erytrosepala Borbas, O. fallax Renner & Rostański [8], Rubus chamaemorus L. [9], Solidago graminifolia (L.) Elliott. [10], Solidago virgaurea L.[10], Pinguicula vulgaris L. [11], Primula veris L. [12], Eryngium planum L. and E. campestre L. [13,14]. Vol. 54 No

3 B. Thiem, J. Budzianowski, M. Wesołowska, L. Ratajczak, M. Morozowska, L. Skrzypczak 160 Ta b l e 1. Studies on in vitro cultures and phytochemical analyses of rare and endangered plants performed by authors species Gentiana cruciata L. in vitro culture callus,shoot cultures, plantlets secondary metabolites chemical class secoiridoid glycosides Gentiana lutea L. shoot cultures, plantlets secoiridoid glycosides Gentiana punctata L. shoot cultures, plantlets secoiridoid glycosides Oenothera ammophila Focke Oenothera erythrosepala Borbas Oenothera fallax Renner em Rostański callus, shoot cultures callus, shoot cultures callus, shoot cultures fatty acids main compounds gentiopicroside amarogentin gentiopicroside amarogentin gentiopicroside amarogentin linoleic, γ-linolenic acids linoleic, γ-linolenic acids linoleic, γ-linolenic acids references Skrzypczak et al.1993 Skrzypczak et al.1993 Skrzypczak et al.1993 Thiem et al Thiem et al Thiem et al Rubus chamaemorus L. shoot cultures polyphenols ellagic acid Thiem 2001, Thiem 2002 Solidago graminifolia (L.) Elliott. Solidago virgaurea L. encapsulated shoot buds Thiem, Krawczyk 2004 micropropagated plants essential oils monoterpene hydrocarbons Kalemba, Thiem 2003 lipophilic flavones under study Budzianowski, Thiem 2008 callus, shoot cultures micropropagated plants chlorogenic acid 3,5- caffeoylquinic acids Skrzypczak et al dicaffeoylquinic acid Ratajczak, Budzianowski 2001; Ratajczak 2007 iridoids, phenylethanoid under study Ratajczak 2007 glycosides Morozowska, Wesołowska 2004 Pinguicula vulgaris L. plantlets flavonoids under study Primula veris L. Eryngium planum L. shoot cultures shoot cultures callus, shoot culture, micropropagated plants lipophilic flavones triterpenoid saponins, phenolic acids zapotin, 2 -methoxyflavone, 3 -methoxyflavone rosmarinic acid saponin complex Budzianowski et al. 2005, 2006 Thiem, Arkuszyńska 2006 Eryngium campestre L. shoot cultures triterpenoid saponins saponin complex Thiem, Wiatrowska 2007 Eryngium alpinum L. shoot cultures triterpenoid saponins saponin complex Thiem (unpubl.) Cultures were initiated from the seeds and sterile explants from 30 day-old seedlings. Clonal propagation procedures have been developed on a Murashige and Skoog [15] medium with different growth plant regulators. Shoot multiplication was achieved through axillary bud cultures. The fidelity of the in vitro-derived plantlets to intact plants was evaluated by a morphological, biochemical, cytogenetical and phytochemical analyses [16]. Stu-

4 Secondary metabolites of in vitro cultures of selected Polish rare and endangered plants dies to ascertain genome size stability following micropropagation was performed on the basis of the nuclear DNA content. Flow cytometric estimation of the nuclear DNA content and check of the ploidy level of different shoot cultures of selected plants has been determined [17,18]. Cytogenetic studies have not shown any variation in micropropagated plants. Protein polymorphism studies of Rubus chamaemorus also confirmed shoot cultures stability [19]. For conservation and in vitro germplasm storage of some rare species, two biotechnological methods were also applied: slow growth techniques for in vitro cold storage and encapsulation of shoot buds (production of artificial seeds). Slow growth of shoot cultures of Rubus chamaemorus (cloudberry) [20] and Solidago graminifolia [16] was developed. Growth reduction was achieved by modifying such parameters as: temperature, light and culture medium. Axillary buds obtained from in vitro shoot culture of cloudberry were encapsulated in a calcium alginate hydrogel [20]. In vitro techniques can be employed for propagation and germplasm storage of rare plant species, after control of shoot cultures fidelity. Therefore, tissue cultures can contribute to the protection of plant genomic resources. In vitro derived plantlets can be used for ex situ germplasm conservation [21]. It is possible to produce a large number of plants, genetically identical to the mother plants, and to introduce them to botanical gardens or to natural habitat now. 161 Phytochemical analyses Plant raw material for phytochemical analyses of the presence of secondary metabolites was obtained from in vitro cultures as well as from micropropagated plants growing in the soil. The methanol extracts were prepared from either dried or fresh plant material. The dried extracts were then fractionated by solvent-solvent extractions to afford chloroform (or dichloromethylene), butanol and water soluble fractions. Exceptionally, also other organic solvents, like ethyl ether and ethyl acetate, were employed for partitioning. Preliminary one-dimensional (1D-TLC) and two-dimensional (2D-TLC) thin-layer chromatography revealed the secondary metabolites pattern for the given plant species. For the isolation and structural elucidation of the detected compounds, the appropriate fractions were separated by a combination of the preparative thin-layer chromatography (PTLC) and column chromatography (CC), using various adsorbents (polyamide, cellulose, silica gel, sephadex LH20) and mobile phases (i.e. solvents mixtures). The structures of the isolated pure compounds were elucidated mainly by means of nuclear magnetic resonance spectroscopy (NMR) using one-dimensional ( 1 H NMR, 13 C NMR) and two-dimensional (HH-COSY, TOCSY, NOESY, HMQC, HMBC) techniques and also by mass spectrometry (MS) and UV spectrophotometry with Vol. 54 No

5 B. Thiem, J. Budzianowski, M. Wesołowska, L. Ratajczak, M. Morozowska, L. Skrzypczak 162 diagnostic shift reagents. in some cases gas chromatography-mass spectrometry (Gc-MS) and hplc were also employed for quantitative analysis. Those investigations of in vitro-derived plants, resulted in the detection of the following secondary metabolites with biological activity. The presence of secoiridoid glucosides (amarogentin and gentiopicroside) in the regenerated in vitro plants, callus tissue and suspension cultures of Gentiana ssp. was determined [22]. The presence of fatty acids, flavonoids and phenolic acids in regenerated plants of some Oenothera species was also determined [8]. From Rubus chamaemorus shoot cultures phenolic acid - ellagic acid was isolated [23]. From the leaves of Primula veris from field cultivation and in vitro cultures ten lipohilic flavones, e.g. zapotin (fig. 1), including four new natural compounds, were isolated and identified [24, 25]. in turn, callus tissues of that species appeared to be able to produce only some metabolites present in the intact plant - saponins but not phenolics (flavonoids) [26]. From the inflorescences of micropropagated Solidago graminifolia lipophilic flavones were obtained and some of them were identified as hydroxy- and methoxy-flavones with various substitution pattern [27]. The composition of the essential oil from aerial parts of clonal propagated plants was also studied [28]. From Pinguicula vulgaris shoot culture iridoids, phenylethanoid glucosides and flavonoids were isolated [29], the structural elucidation of which is underway. Figure 1. Two-dimensional magnetic resonance spectrum (2d nmr) (hmqc) of the lipophilic flavone (zapotin) from Primula veris The presence of triterpene saponins, flavonoids and phenolic acids in shoot cultures, callus and biomass of cells from suspension culture of Eryngium planum has been determined [30].

6 Secondary metabolites of in vitro cultures of selected Polish rare and endangered plants Some quantitative analyses were also carried out. Quantitative determination of secoiridoids in micropropagated Gentiana cruciata was made by TLC-densitometry [22]. The content of fatty acids, especially the unsaturated γ-linolenic acid in seeds of micropropagated Oenothera ammophila, O. fallax and O. erythtrosepala was determined using the gas chromatography (GC) method [8]. The quantitative analysis of ellagic acid in in vitro shoot cultures of Rubus chamaemorus and in leaves of intact plants was determined by high performance liquid chromatography (HPLC) [31], [32]. The HPLC analysis of saponins and rosmarinic acid in in vitro cultures of Eryngium planum were employed for quantitative studies [30]. In general, the level of determined compounds in in vitro-derived material was comparable to that of intact plants. The results of our studies showed that those rare plants contain pharmaceutically promising compounds with wide spectrum of activities, eg. anticancer (ellagic acid, lipophilic flavones, especially zapotin), antioxidative (rosmarinic acid, ellagic acid, -linolenic acid), antiinflammatory effects (γ-linolenic acid), anti-coughing (saponins, cinnamonic acids derivatives) and for the treatment of gastrointestinal tract diseases (gentiopicroside). 163 Conclusions Our studies revealed that biotechnology gives an opportunity to carry out phytochemical investigations of rare, vulnerable or endangered plants without collecting them from natural localities. Moreover, rare and endangered plants in form of micropropagated plants, callus tissues and biomass of cells can provide an appropriate raw material not only for phytochemical analyses, but also for pharmaceutical purposes (fig. 2). Vol. 54 No

7 B. Thiem, J. Budzianowski, M. Wesołowska, L. Ratajczak, M. Morozowska, L. Skrzypczak 164 A1 A2 B1 B2 C1a C1b C2 D1 D2 Figure 2. In vitro cultures (A1 D1) shoot cultures (A1, B1, D1), callus tissue (C1a), cell suspension (C1b) and thin-layer chromatograms (A2 D2) of extracts from Rubus chamaemorus (A), Pinguicula vulgaris (B), Eryngium planum (C), Primula veris (D)

8 Secondary metabolites of in vitro cultures of selected Polish rare and endangered plants 165 References 1. Kukułczanka K. Kultury in vitro w zachowaniu gatunków rzadkich i ginących. Wiad Bot Biul Ogrodów Bot Muzeów i Zbiorów 1987; 31: Kukułczanka K. Cząstka B. Rozmnażanie wybranych gatunków Droseraceae i utworzenie banku genów w kulturze in vitro. Prace Ogrodu Botanicznego PAN 1991; 1: Rybczyński J, Mikuła A. Biotechnologia w zachowaniu różnorodności flory Polski. Materiały ogólnopolskiej konferencji naukowej Rzadkie, ginące i reliktowe gatunki roślin i grzybów. Kraków Abstracts 2006: (with English summary). 4. Łojkowska E. Mikrorozmnażanie roślin chronionych. X Ogólnopolska Konferencja Kultur In Vitro i Biotechnologii Roślin, Bydgoszcz, , 2003, Abstracts. 19.(with English summary). 5. Ashmore SE. Status report on the development and application of in vitro techniques for the conservation and use of plant genetic resources. International Plant Genetic Resources Institute; Rome, Italy, 1997: Puchalski J. Banki genów w zachowaniu roślin rzadkich i zagrożonych. Biuletyn Ogrodów Botanicznych 2000; 9: Skrzypczak L, Wesołowska M, Skrzypczak E. Gentiana species: In vitro culture, regeneration, and production of secoiridoid glucosides. In: Bajaj YPS (ed.). Biotechnology in Agriculture and Forestry, Vol. 21, Medicinal and Aromatic Plants IV. Springer, Berlin-Heidelberg 1993: Thiem B, Skrzypczak L, Lamer-Zarawska E. Micropropagation of selected Oenothera species and preliminary studies on their secondary metabolites. Acta Soc Bot Polon 1999; 68: Thiem B. Micropropagation of cloudberry (Rubus chamaemorus L.) by initiation of axillary shoots. Acta Soc Bot Polon 2001; 70: Skrzypczak L, Wesołowska M, Thiem B, Budzianowski J. Solidago L. species (Goldenrod): In vitro regeneration and biologically active secondary metabolites. In: Bajaj YPS (ed.). Biotechnology in Agriculture and Forestry, Vol. 43, Medicinal and Aromatic Plants XI. Springer, Berlin Heidelberg 1999: Ratajczak L. Metabolity wtórne w Pinguicula vulgaris L. z kultur in vitro. Rozprawa doktorska (PhD dissertation, in Polish language with English summary), Wydział Farmaceutyczny, Uniwersytet Medyczny im. Karola Marcinkowskiego w Poznaniu, Morozowska M, Wesołowska M. In vitro clonal propagation of Primula veris L. and preliminary phytochemical analysis. Acta Biol Cracov Bot, 2004; 46: Thiem B, Wesołowska M, Skrzypczak L. Eryngium planum L. w kulturze in vitro. XI Ogólnopolska Konferencja Kultur In Vitro i Biotechnologii Roślin, Kultury in vitro podstawą biotechnologii roślin. Międzyzdroje , Abstracts 2006: 89 (with English summary). 14. Thiem B, Wiatrowska I. Mikrorozmnażanie Eryngium campestre L. 54 Zjazd PTB Szczecin Streszczenia referatów i plakatów: Murashige R, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962; 15: Thiem B. Techniki biotechnologiczne zastosowane do oceny i zachowania wybranych cech roślin leczniczych zwiększających ich wykorzystanie w fitoterapii. Praca habilitacyjna (in Polish language), Wydział Farmaceutyczny Uniwersytet Medyczny im. Karola Marcinkowskiego w Poznaniu; Thiem B, Śliwińska E. Flow cytometric analysis of nuclear DNA content in cloudberry (Rubus chamaemorus L.) in vitro cultures. Plant Sci 2003; 164: Śliwińska E, Thiem B. Genome size stability in six medicinal plant species propagated in vitro. Biologia Plantarum 2007; 51: Thiem B, Kalinowski A. Comparison of protein patterns after two-dimensional gel electrophoresis from leaves of in vitro cultures and seedlings of Rubus chamaemorus L. Acta Soc Bot Polon 2002; 71: Thiem B. In vitro culture techniques in conservation of Rubus chamaemorus L. Acta Biol Cracov Bot 2002; 44: Engelmann F. Brief overview of IPGRIs research activities on in vitro conservation of plant species. Botanic Gardens Micropropagation News 1995; 2: Krupińska A, Segiet-Kujawa E, Skrzypczak L, Ellnain-Wojtaszek M. Quantitative determination of amarogentin by TLC - densitometry. Sci. Pharm. 1991; 59:135. Vol. 54 No

9 B. Thiem, J. Budzianowski, M. Wesołowska, L. Ratajczak, M. Morozowska, L. Skrzypczak Thiem B, Krawczyk A, Budzianowski J. Ellagic acid in the intact plants and in vitro propagated Rubus chamaemorus L. The 3 rd International Symposium on Chromatography of Natural Products June 2002, Lublin Kazimierz Dolny, Poland, Abstract 2002: Budzianowski J, Morozowska M, Wesołowska M. Lipophilic flavones of Primula veris L. from field cultivation and in vitro culture. Phytochemistry 2005; 66: Budzianowski J, Morozowska M, Wesołowska M. Comparative study of lipophilic flavones in Primula veris L. from natural localities, field cultivation and in vitro culture. 5 th International Symposium on Chromatography of Natural Products (ISCNP) (The Application of Chromatographic Methods in Phytochemical and Biomedical Analysis), Lublin June 2006: Wesołowska M, Morozowska M, Kosińska A. Tkanka kalusowa Primula veris L. z kultur in vitro. XI Ogólnopolska Konferencja Kultur In Vitro i Biotechnologii Roślin. Kultury in vitro podstawą biotechnologii roślin. Międzyzdroje , Abstracts 2006:203 (with English summary). 27. Budzianowski J, Thiem B. Further lipophilic flavonoids of Solidago graminifolia (L.) Elliot. 6 th International Symposium on Chromatography of Natural Products (ISCNP) The application of chromatographic methods in phytochemical & biomedical analysis. Lublin (Poland), June Abstract Book: Kalemba D, Thiem B. Constituents of the essential oils of four micropropagated Solidago species. Flav Fragr J 2004; 19: Ratajczak L, Budzianowski J. Wstępna analiza związków fenolowych Pinguicula vulgaris L. z kultur in vitro. 52 Zjazd Polskiego Towarzystwa Botanicznego, Poznań Abstracts 2001: Thiem B, Arkuszyńska A. Metabolity wtórne w kulturze zawiesinowej Eryngium planum L. VIII Ogólnopolskie Akademickie Seminarium Studentów Biotechnologii, Kraków , Abstracts: Krawczyk A, Thiem B, Szkudlarek M. High-performance liquid chromatography of ellagic acid in leaves of Rubus chamaemorus L. Chem. Anal Warsaw 2003; 48: Thiem B, Krawczyk A. Ellagic acid in in vitro cultures of Rubus chamaemorus L. Herba Pol 2004; 49: Metabolity wtórne kultur in vitro wybranych polskich roślin rzadkich i ginących Barbara Thiem 1*, J. Budzianowski 1, M. Wesołowska 1, L. Ratajczak 2, M. Morozowska 3, L. Skrzypczak 1 1 Katedra Botaniki Farmaceutycznej i Biotechnologii Roślin Uniwersytet Medyczny im. Karola Marcinkowskiego ul. Św. Marii Magdaleny Poznań 2 Katedra i Zakład Naturalnych Surowców Leczniczych i Kosmetycznych Uniwersytet Medyczny im. Karola Marcinkowskiego ul. Mazowiecka Poznań 3 Wydział Botaniki Uniwersytet Przyrodniczy im. Augusta Cieszkowskiego ul. Wojska Polskiego 71C Poznań *autor, do którego należy kierować korespondencję: bthiem@ump.edu.pl

10 Secondary metabolites of in vitro cultures of selected Polish rare and endangered plants 167 S t r e s z c z e n i e Przedstawiono krótki przegląd prac dotyczących analiz fitochemicznych kultur in vitro wybranych rzadkich i zagrożonych polskich roślin. Analizowane metabolity wtórne występujące w badanych gatunkach są odpowiedzialne za właściwości lecznicze surowców. Związki fenolowe (flawonoidy i kwasy fenolowe), sekoirydoidy, saponiny i kwasy tłuszczowe zostały zbadane w roślinach uzyskanych w drodze mikrorozmnażania, kulturach pędowych i tkance kalusowej 11 gatunków roślin z rodzajów Gentiana L., Oenothera L., Rubus L., Solidago L., Pinguicula L., Primula L. i Eryngium L. Badane gatunki są roślinami leczniczymi albo roślinami wykorzystywanymi w medycynie ludowej, w których z reguły za właściwości lecznicze są odpowiedzialne metabolity wtórne. Badania wykazały, że te rośliny zawierają cenne związki, które mogą znaleźć zastosowanie farmaceutyczne. Dlatego biotechnologia roślin może być wykorzystywana dla szybkiego mnożenia rzadkich, a także zagrożonych roślin, a zregenerowane rośliny mogą zostać użyte jako źródło tych rzadkich gatunków leczniczych. Słowa kluczowe: kultury in vitro, mikrorozmnażanie, wtórne metabolity, analizy fitochemiczne Vol. 54 No