rhodiola kirilowii – the present status and perspectives...

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Rhodiola kirilowii – the present status and perspectives of medicinal use Part I. In vivo and in vitro cultivation as well as phytochemical investigations of extracts of roots and callus tissues AnnA KRAjeWSKA-PAtAn 1 *, MIRoSłAWA FuRMAnoWA 2 , MARIolA DRegeR 1 , AlInA MścISZ, 1 SeBAStIAn MIelcAReK 1 , MAłgoRZAtA KAnIA 1 , WAlDeMAR BuchWAlD 1 , MAReK BARAnIAK 1 , AgnIeSZKA PIetRoSIuK 2 , MonIKA Zych 2 , MonIKA KARASIeWIcZ 1 , AnnA BogAcZ 1 , RADoSłAW KujAWSKI 1 , PRZeMySłAW M. MRoZIKIeWIcZ 1 1 Research Institute of Medicinal Plants libelta 27 61-707 Poznań, Poland 2 Department of Biology and Pharmaceutical Botany Medical university Banacha 1 02-097 Warsaw, Poland *corresponding author: tel.: 48 61 6659540, fax: 48 61 6659551, e-mail: [email protected] Summary Rhodiola kirilowii (Regel) Maxim (Crassulaceae family) is used in traditional east Asian medicine, mainly in china, to prevent damages due to hypoxic environment of high altitude. on the basis of own achievements and wide review of the published articles the authors describe the status of field cultivation as well as phytochemical and biotechnological investigations carried out on R. kirilowii (callus tissue cultures, micropropagation in vitro, somatic seeds pro- duction, hairy roots cultures). the identified chemical constituents of the extracts of roots and callus tissues of R. kirilowii are presented and divided according to chemical groups. Key words: Rhodiola kirilowii, traditional medicine, hypoxia, field cultivation, in vitro cultures, micropropagation, hairy roots, somatic seeds, phytochemical investigations, salidroside, rosavins Rhodiola sp. – traditional medicinal plant Rhodiola plants belong to herbs used in traditional medicine in china, in Russia and in northern europe. the best known is Rhodiola rosea l. – goldenroot. R. rosea grows

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Page 1: Rhodiola kirilowii – the present status and perspectives ...herbapolonica.pl/magazines-files/5227073-12_Rhodiola.pdf · Rhodiola kirilowii – the present status and perspectives

140A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

Rhodiola kirilowii – the present status and perspectives of medicinal usePart I. In vivo and in vitro cultivation as well as phytochemical investigations of extracts of roots and callus tissues

AnnA KRAjeWSKA-PAtAn1*, MIRoSłAWA FuRMAnoWA2, MARIolA DRegeR1, AlInA MścISZ,1 SeBAStIAn MIelcAReK1, MAłgoRZAtA KAnIA1, WAlDeMAR BuchWAlD1, MAReK BARAnIAK1, AgnIeSZKA PIetRoSIuK2, MonIKA Zych2, MonIKA KARASIeWIcZ1, AnnA BogAcZ1, RADoSłAW KujAWSKI1, PRZeMySłAW M. MRoZIKIeWIcZ1

1Research Institute of Medicinal Plantslibelta 2761-707 Poznań, Poland2Department of Biology and Pharmaceutical BotanyMedical universityBanacha 102-097 Warsaw, Poland

*corresponding author: tel.: 48 61 6659540, fax: 48 61 6659551, e-mail: [email protected]

S u m m a r y

Rhodiola kirilowii (Regel) Maxim (Crassulaceae family) is used in traditional east Asian medicine, mainly in china, to prevent damages due to hypoxic environment of high altitude. on the basis of own achievements and wide review of the published articles the authors describe the status of field cultivation as well as phytochemical and biotechnological investigations carried out on R. kirilowii (callus tissue cultures, micropropagation in vitro, somatic seeds pro-duction, hairy roots cultures). the identified chemical constituents of the extracts of roots and callus tissues of R. kirilowii are presented and divided according to chemical groups.

Key words: Rhodiola kirilowii, traditional medicine, hypoxia, field cultivation, in vitro cultures, micropropagation, hairy roots, somatic seeds, phytochemical investigations, salidroside, rosavins

Rhodiola sp. – traditional medicinal plant

Rhodiola plants belong to herbs used in traditional medicine in china, in Russia and in northern europe. the best known is Rhodiola rosea l. – goldenroot. R. rosea grows

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in many places in europe, in opposite to R. kirilowii, growing widely only in Asia. tor the first time, R. rosea was described by Dioscorides, the greek physician, in De Materia Medica in 77 cA. In the 17th century the medical properties of Rhodiola were cited by linne in his elaborates on Scandinavian flora: in the Flora lapponica [1], the Flora Su-ecica [2] and in the Materia Medica holmiae [3] (fig. 1). linne described Rhodiola rosea as a remedium for treatment rupture, vaginal discharges, headache and hysteria [3]. In Asia, especially in china and tibetan traditional medicine as well as in Siberian folk medicine Rhodiola was used as a plant adapting a human body to disadvantageous en-vironmental conditions and improving physician and mental abilities. other disorders treated in ethnomedicine with Rhodiola are as follows: fatigue, depression, anemia, alleviate nervous disorders. It is also used in treatment of high altitude sickness and to enhance fertility. In china divers, astronauts, pilots and mountaineers use Rhodiola to enhance their abilities. the positive properties of extracts from Rhodiola plants in adaptation to environmental conditions caused that nowadays R. rosea extracts become serving as constituents of modern medicines of plant origin used to enhance physician and human abilities, also in european countries.

Botanical characteristics

Rhodiola genus (Crassulaceae family) consists of more than 50 species. For the first time Rhodiola kirilowii (Regel) Maxim. was described under this name in 1859 in Russia in the article published by Imperial Academy of Science in Saint Pe-tersburg in the journal entitled “Mémoires présentés á l’Académie Impériale des Sciences de St.-Petersburg par Divers Savants et lux dans ses assemblées” (accor-ding to Flora of china [4]). other synonyms of Rhodiola kirilowii are Sedum kiriolwii Regel and Sedum elongatum Kar. [4, 5]. R. kirilowii belongs to Linearifoliae A. Bor. group of Rhodiola genus [5]. In chinese is called xia ye hong jing tian [4].

R. kirilowii, a perennial plant, has erect, thick root and rhizomes with lanceolate or ovate scalelike leaves. the stems are up to 60 (max. 90) cm high, density leafy. leaves are alternate or subverticillate, linear to linear-lanceolate, on the margin sparsely serrulate. Flowers are bi- or unisexual 4- or 5-merous, with green, gre-enish yellow or red petals. Seeds are oblong-lanceolate, ca. 1.5 mm. R. kirilowii flowers at May-September. the plant grows in mountains at the altitude of 2000-5600 m in Asia: mainly tien Shan, Altaj, Pamir. the natural sites of R. kirilowii are forest margins, grassy slopes, often in partial shade [4, 5].

Rhodiola KiRilowii in field cultivation and in micropropagation in in vitRo conditions

the investigations on R. kirilowii in Poland started about 2000 and were carried out in cooperation in two laboratories: Department of Biology and Pharmaceutical

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142A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

a.

b.

c. Figure 1. Rhodiola sp. in caroli linnaei publications – figures obtained by kindness of Anders Backlund, Division of Pharmacognosy, Department of Medicinal chemistry, uppsala universitya. linnaei c. Flora Suecica, 1745 [2]b. linnaei c. Materia Medica holmiae, 1749 [3]c. linnaei c. Flora lapponica, 1737 [1]

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Botany, Warsaw Medical university and the Research Institute of Medicinal Plants (RIMP), Poznań. these searching were supported by grants from Polish commit-tee for Scientific Research (no PBZ-KBn-092/Po5/2003) and from the Ministry of Science and high education (project no n405 025 32/1687).

Plants were grown in the RIMP garden of Medicinal Plants in Plewiska near Poznań (fig. 2). on the basis of these five-year investigations the instruction for the field cultivation of this species was elaborated [RIMP – unpublished data]. the obtained raw plant material was investigated for chemical constituents and served as the start material for callus tissue cultures. the getting results proved that the field cultivation of this species of Asian origin is possible in Polish climate. Phytochemical investigations of the obtained root extracts allowed to determine the main chemical constituents, widening the known chemical spectrum of the compounds. the results of these investigations are summarized in the chapter about phytochemical investigations of roots of R. Kirilowii.

Following the above-mentioned study on field cultivation the investigators from RIMP and Warsaw Medical university searched the micropropagation of R. kirilowii in in vitro conditions [6]. Micropropagation of R. kirilowii was studied for the first time by chinese group in 2004 (li et al. [7]), but their achievements were not widely distributed due to language barrier. Polish investigators from Warsaw Medical university elaborated the simple method of plants propagation from sterile seedlings connected with somatic seeds production [6, 8]. the group from Warsaw Medical university and the group from RIMP searched the encapsulation of axillary buds and differentiating callus tissue of R. Kirilowii using sodium alginate mixed with pure Murashige-Skoog medium [9] or medium supplemented with growth regulators. After encapsulation the somatic seeds were stored at 4ºc for 1 to 15 weeks and then their viability was tested on MS medium. the results showed that after six weeks of preservation 100% of somatic seeds were able to develop into plantlets. Authors suggest that not only axillary buds could be encapsulated. the small pieces of differentiating callus tissue can be used in somatic seeds production as well [6].

phytochemical investigations of R. RoSea roots, the Best known medicinal plant from RhoDIoLa sp.

the modern chemical investigation on Rhodiola sp. started in the seventies of 20th century. It allows to determine the main active constituents of the extracts: salidro-side (glycoside of p-tyrosole) [10, 11], occurring in many Rhodiola species, and ro-savins (phenylopropanoids derivatived from cinnamyl alcohol) treated like specific compounds of R. rosea [12]. goldenroot belongs to more often investigated Rhodiola plants and about 100 compounds have been isolated and identificated from roots and underground parts of this plant: rosavins [12], p-tyrosol, salidroside [10, 11], flavonoids [13], phytosterols (daucosterol and β-sitosterol) [14], chlorogenic acid, caffeic acid, gallic acid, hydroxycinnamic acid, coumaric acid [15, 16], cyanogenic glycoside – lotaustralin [17], essential oil [18], fatty acids [19].

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144A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

a. b.

c. d.Figure 2. Rhodiola kirilowii (Regel.) Maxim. cultivated in the garden of Medicinal Plants, Research Institute of Medicinal Plants in Poznana, b. 5-year-old plantationc. 5-year-old flowering plantd. root of 5-year-old plant

phytochemical investigations of R. KiRilowii roots

on the contrary to relatively well investigated extracts from R. rosea, R. kirilowii is searched mainly in china and the obtained results are not widely known, due to the fact that most of them are published in chinese. According to Russian in-vestigations carried out on Rhodiola sp. in the seventies of 20th century, R. kirilowii

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contains: salidroside, p-tyrosole, gallic acid, coumarins esculetin and umbellifero-ne, flavonoid herbacitrin [20]. chinese searchers communicated that R. Kirilowii contains bergenin, identified by spectral methods by Zhang et al. [21]. β-sitosterol in R. Kirilowii was described for the first time in 1992 by Kang et al. [22]. the other phytosterol – daucosterol was found in R. Kirilowii rhizome by Peng and cowor-kers in 1994 [23]. they also identified lotaustralin, an toxic cyanogenic glycoside, in this plant material [23]. this compound was searched by gc methods by Kang et al. – according to their opinion, R. Kirilowii was the plant of the highest content of lotaustralin in ten investigated Rhodiola species [24, 25].

In recent years the growing interest in R. Kirilowii searchings is observed with the use of modern identification and quanitative compounds analysis methods. the examples are cui et al. investigations [26] carried out on R. Kirilowii and R. crenulata by capillary zone electrophoresis (cZe). this team elaborated the rapid method of simultaneous determination of p-tyrosol and salidroside in plant mate-rial with suggestion to use this method for bulky samples and quality control in pharmaceutical plants [26].

the results of first chemical investigations carried out in the Department of Biology and Pharmaceutical Botany of Warsaw Medical university and in the Rese-arch Institute of Medicinal Plants (RIMP) in Poznań on the material grown in the RIMP garden of Medicinal Plants in Plewiska near Poznan were described in 2002 [27]. In next years the self-elaborated hPlc method allowed to determine next compounds in the hydroalcoholic extract of plant material from field cultivation: gallic acid – in higher concentration than in R. rosea [28-31], epigallocatechin gal-late (also in the higher concentration than in R. rosea) [28, 29, 32], tannins [28, 31], p-tyrosol, salidroside and – for the first time in this species – rosavins [31]. A new compounds, namely rhodiocyanoside A (cyanogenic glycoside), arbutin and fructopyrano-(1-4)-glucopyranose have been found recently by Wiedenfeld et al. [33] in the root extract from the plants cultivated in Poland (in RIMP).

testing the extract activity against chronic hepatitis c virus the group of chinese investigators has found for the first time some compounds in the extract from R. kirilowii: 3,3’-digalloylproprodelphinidin B2 (rhodisin), 3,3’-digalloylprocyanidin B2, (-)-epicatechin-3-o-gallate (ecg), (-)-epigallocatechin, (-)-epicatechin, luteolin, trice-tin, rhodiolinozide [34]. they also confirmed, that in R. kirilowii extracts (-)-epigallo-catechin-3-o-gallate (egcg), gallic acid, tyrosol and salidroside are present [34].

Searching the anti-tuberculosis activity, another group of chinese investiga-tors separated and identified twelve compounds from R. kirilowii extracts. Among them a few compounds were isolated from this species for the first time, namely trans-hydroxycinnamic acic, hexyl-β-glucopyranoside, rhodiolgin, isolariciresinol-9-o-β-glucopyranoside, rhodiooctanoside, sacranoside B, geranyl-β-glucopyrano-side and neryl-β-glucopyranoside. Moreover, they isolated β-sitosterol, p-tyrosol, gallic acid and -epigallocatechin gallate [35].

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146A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

the constituents identified or searched in the roots of R. kirilowii are summari-zed in table 1. the structures of most of them are presented in figures 3–6.

ta b l e 1 .

Identified phytochemical constituents of Rhodiola Kirilowii plants

class or group of compounds

compound name identified or searched in plant’s extract by

phenylethanoids

p-tyrosol

salidroside

Krasnov et al. [20], Kang et al. [22], Peng et al. [23], cui et al. [26], Krajewska-Patan et al. [31], Zuo et al. [34], Wong et al. [35]Krasnov et al. [20], Kang et al. [22], Peng et al. [23], cui et al. [26], Krajewska-Patan et al. [31], Wiedenfeld et al. [33], Zuo et al. [34]

phenylopropanoidsrosavin, rosin, rosarintrans-hydroxycinnamic acid

Krajewska-Patan et al. [31]Wong et al. [35]

flavonoids

luteolinherbacitrintricetinrhodiolginepigallocatechin gallate

(-)-epicatechin-3-o-gallate (ecg)(-)-epigallocatechin(-)-epicatechin

Zuo et al. [34]Krasnov et al. [20] Zuo et al. [34]Wong et al. [35]Mścisz et al. [28], Mielcarek et al. [30], Buchwald et al. [29], Krajewska-Patan et al. [31], Wiedenfeld et al. [33], Zuo et al. [34], Wong et al. [35]

Zuo et al. [34]Zuo et al. [34]Zuo et al. [34]

hydroxycoumarins esculetin, umbelliferone Krasnov et al. [20]

isocoumarins bergenin Zhang [21]

phenolic acidsgallic acid Krasnov et al. [20], Mścisz et al. [28], Mielcarek et al. [30],

Buchwald et al. [29], Krajewska-Patan et al. [31], Zuo et al. [34], Wong et al. [35]

phytosterolsβ-sitosteroldaukosterol

Kang et al. [22]Wong et al. [31]

tannins3,3’-digalloyl-proprodelphinidin B2 (rhodisin)3,3’-digalloyl-procyanidin B2

Zuo et al. [34]

Zuo et al. [34]

cyanogenic glycosideslotaustralin

rhodiocyanoside A

Peng et al. [23], Kang and Wang [24], Kang et al. [25], Wiedenfeld et al. [33]Wiedenfeld et al. [33]

phenols arbutin Wiedenfeld et al. [33]

lignans isolariciresinol-9-o-β-glucopyranoside Wong et al. [35]

phenolic ketones rhodiolinozide Zuo et al. [34]

oligoglycosides rhodiooctanoside Wong et al. [35]

oligosaccharidessucrosefructopyrano-(1-4)-glucopyranosehexyl-β-glucopyranoside

Peng et al. [23]Wiedenfeld et al. [33]Wong et al. [35]

terpenoidssacranoside Bgeranyl-β-glucopyranosideneryl-β-gluco-pyranoside

Wong et al. [35]Wong et al. [35]Wong et al. [35]

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HO

OH

OHO

HO

HO

O

OH

OH

a) tyrosol b) salidroside

O

OHO

HO

HO

O

H2CO

OH

HO

OH

OHOH2C

HOHO

HO

O

c) rosavin d) rosin

O

OH

OHHO

OO

HOHO

HO

O

H2C

OH

COOH

e) rosarin f) trans-4-hydroxycinnamic acid

HO COOH

H

HOH OH

H

O

O

OH

OH

OH

HO

COOH

g) chlorogenic acid h) caffeic acid

Figure 3. the chemical structures of roots and/or biomass components identified in R. kirilowii – phenylethanoids and phenylopropanoids.structures a), f) – h) according to Phytochemical Dictionary [36]structure b) according to Kir’anov et al. [10]structures c) – e) according to Zapesochnaya and Kurkin [12]

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148A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

OHO

OH

OH

OH

O

OHO

OH

OH

OH

O

OH

a) luteolin b) tricetin

OHOH

H

O

OHOH

OH

O

OH

OH

OH

OH

c) epigallocatechin 3-gallate

OHOH

H

O

OHOH

O

OH

OH

OH

OH

OHO

OHOH

OH

OH

OH

d) epicatechin-3-gallate e) epigallocatechin

OHO

OH

OH

OH

H

H

OH

f) epicatechin

Figure 4. the chemical structures of roots and/or biomass components identified in R. kirilowii – flavonoids.structures a) and b) according to Phytochemical Dictionary [36]structures c) – f) according to Zuo et al. [34]

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Rhodiola kirilowii – the present status and perspectives of medicinal use. Part I

O O

HO

HO O OHO

a) esculetin b) umbelliferone

OH

CH3O

HOO

O

HO

H CH2OH

OHH

HHOH

COOH

OH

OH

HO

c) bergenin d) gallic acid

HO

H

H

H

H

H H

e) beta sitosterol

OHO

OHOH

OH

OH

OR

HO

OH

O

OR

OH

OH

OH

R=

CO

OH

OH

HO

OHO

OHOH

OH

OR

HO

OH

O

OR

OH

OH

R=

CO

OH

OH

HO

f) rhodisin g) digalloylprocyanidin B2

Figure 5. the chemical structures of identified in R. kirilowii roots and/or biomass components – hydroxycoumarins, isocoumarins, phenolic acids, tannins.structures a) – e) according to Phytochemical Dictionary [36]structures f) and g) according to Zuo et al. [34]

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150A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

O

OH

HOHO

HO

O

CN

O

OH

HOHO

HO

O

CN

a) lotaustralin b) rhodiocyanoside A

O

OH

HOHO

HO

OH

O

COCH3

H3CO

OH

OGlc

Glc=beta D glucopyranosyl

c.) arbutin d) rhodiolinozide

O

O

O

CH2OH

CH2OHOHHO

CH2OHHOHO

OH

O

OH

OH

OH

OH

O

O

OH

OH

OH

OH

e) sucrose f) fructopyrano-(1-4)-glucopyranose

Figure 6. the chemical structures of identified in R. kirilowii roots and/or biomass components – cyanogenic glycosides, phenols, phenolic ketones, oligosaccharides. structures a), c), e) according to Phytochemical Dictionary [36]structures b) and f) according to Wiedenfeld et al. [33]structure d) according to Zuo et al. [34]

the main active compounds of the hydroalcoholic extract of roots of R. kirilowii can be detected by hPlc method elaborated in the Research Institute of Medicinal Plants in cooperation with Department of Biology and Pharmaceutical Botany of Warsaw Medical university (fig. 7).

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Figure 7. hPlc chromatogram of the extract (50% etoh) from R. Kirilowii roots

plant tissue cultures in vitRo of R. KiRilowii and phytochemical investigations of Biomass

Research communications about R. kirilowii tissue cultures started to appear in 2004: li and coworkers [37] described a method of rapid micropropagation of R. Kirilowii. Furthermore, Krajewska-Patan and coworkers from Research Institute of Medicinal Plants in Poznan described their investigations on callus elicititation methods [38]. the group from RIMP has obtained callus tissue from cotyledon, hypocotyle, epicotyle and roots of sterile seedlings on Murashige-Skoog’s me-dium [9] supplemented with benzyloaminopurine (BA), naphtylacetic acid (nAA), adenine hydrochloride or dichlorophenxyacetic acid (2,4-D) [38, 39]. In these inve-stigations the conditions of callus culture were determined and optimized (fig. 8). Authors measured the tissue growth dynamics and growth parameters (fresh and dry weight, percent of dried weights, growth rate). According to these investi-gations, the fresh weights of callus obtained from cotyledons reached maximum values between days 35 and 42 of culture and approximated 14–21 g per one cul-ture cultivated in 200 ml jar). It means that the fresh weights increased 5–8 times during the period of culture [38, 39].

Salidroside epigallocatechin gallate

gallic acid RosarinRosin

Rosavin

chlorogenic acid caffeic acid

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152A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

a) b)Figure 8. R. kirilowii – callus tissue cultures according to Krajewska-Patan et al. [38] and Mrozikiewicz et al. [39]a) callus originated from hypocotyl cultivated on MS medium supplemented with nAA, BA and

adenine chlorideb) callus cultivated in dark condition on MS medium supplemented with 2,4-D

the data shown by Mrozikiewicz and coworkers from the Research Institute of Medicinal Plants in Poznań published in 2006 was the first report on phyto-chemical constituents of callus tissue from R. Kirilowii [39]. the callus tissue from cotyledon was able to synthesize the same biological compounds as those of in vivo plants but the contents of them were much lower [RIMP, unpublished data]. the authors identified some active components of extracts from callus tissue, na-mely salidroside, p-tyrosol, tannins, gallic acid, chlorogenic acid, caffeic acid [32, 39] and tested their contents in vitro during seven-week vegetation period. the content of salidroside varied from 0.08 mg/100 g to 1.56 mg/100 g of dry weight (it is worth expressing that salidroside content decreased rapidly between fourth and fifth weeks), p-tyrosol: 0.06-0.17 mg/100 g, tannins (expressed as pyrogallol) 0.20-0.73%, gallic acid 20.17–38.18 mg/100 g, caffeic acid 6.65–9.94 mg/100 g, chlorogenic acid up to 2.80 mg/100 g of dry weight [32]. lotaustralin was not detected in the callus tissue. the callus tissue obtained from hypocotyle and from radicula of seedlings were able to synthesize the same components as described above: salidroside up to 3.29 mg/100 g, chlorogenic acid (up to 28.47 mg/100 g), p-tyrosol, tannins, phenolic acids, epigallocatechin gallate and rosavins, but their contents were lower than in the intact plant [32]. the latest two components were determined for the first time in callus tissue of R. Kirilowii [31].

Suspension culture of R. kirilowii was also tested but the contents of active components were lower than in callus tissue cultivated on solid medium [31].

the constituents identified or searched in in vitro cultivated plant material are summarizied in table 2. the structures of most of components of biomass of R. kirilowii are presented in figures 4–6.

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ta b l e 2 .

Identified phytochemical constituents of callus of Rhodiola kirilowii

group and names of the compounds identified or searched in callus extract by

phenylethanoids – p-tyrosol, salidroside

li et al. [37], Krajewska-Patan et al. [31, 32]

phenylopropanoids – rosavin, rosin, rosarin, chlorogenic acid, caffeic acid

Krajewska-Patan et al. [31, 32, 40]

phenolic acids – gallic acid Krajewska-Patan et al. [31]

tannins Krajewska-Patan et al. [31, 32]

flavonoids – epigallocatechin gallate Krajewska-Patan et al. [32]

the process of biotransformation of exogenous added precursors in callus tissue of R. kirilowii was tested by Krajewska-Patan and coworkers in 2008 [40]. callus tissue supplemented with p-tyrosol produced almost 70 times more of sa-lidroside than the intact plants cultivated in Polish climatic conditions – up to 1100 mg/100 g. After supplementation with cinnamyl alcohol this type of callus tissue produced also a big amount of rosavins (mainly rosin) – up to 1200mg/100g [40]. this was the first report – according to the known literature – on artificial addition of precursors to R. kirilowii callus tissue [40].

this group of investigator from RIMP and Warsaw Medical Academy tested also the process of elicitation using yeast extract (ye) and methyl jasmonate (jAMe) in 1–10 mg/culture and 100 μM–1 mM/culture concentrations, respectively. they tested biomass changes and the influence on the content of bioactive components [38].

investigations on the transformed hairy roots of R. KiRilowii

the research team from the Department of Biology and Pharmaceutical Botany of Warsaw Medical Academy elaborated the method of obtaining the hairy roots of R. kirilowii by infection of the plantlets with agrobacterium rhizogenes lBA 9402 (with using acetosyringone - 2 mg/l - in the incubation medium). It was the first report on transformation process of this species using agrobacterium rhizogenes. Moreover, according to known literature it was the first obtaining of the hairy roots in the Rhodiola genus [41]. this achievement is worth of notice because transformation of R. kirilowii plants using bacterial vector is very difficult. hairy roots cultures grew the best on hormone free DcR medium. the best plantlet fragments to obtain hairy roots are internodal segments of shoots. the process of transformation was confirmed in the Research Institute of Medicinal Plants ge-netic laboratory using polymerase chain reaction (PcR). DnA analysis showed that rol B gene and aux 1 gene from Ri plasmid of a. rhizogenes were incorporated into the plant genome of R. kirilowii [41]. the phytochemical analysis and biological activity of the obtained hairy roots are in progress.

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154A. Krajewska-Patan, M. Furmanowa, M. Dreger, A. Mścisz, S. Mielcarek, M. Kania, W. Buchwald, M. Baraniak, A. Pietrosiuk,

M. Zych, M. Karasiewicz, A. Bogacz, R, Kujawski, PM. Mrozikiewicz

conclusions

the above presented agricultural investigations showed that it is possible to obtain valuable plant material in Polish climatic conditions. the results of phy-tochemical investigations carried out mainly in china and in Poland allowed to find certain chemical constituents of the extracts, but precise determination of chemical spectrum of that plant is needed. Above-mentioned phytochemical inve-stigations show that chemical constituents of extracts from R. kirilowii roots are in most cases similar to the components isolated from R. rosea roots, with excep-tion of the content of rosavins which presence in some plant material of definite origin was not detected. the future investigations may be allow to answer on the question whether production of rosavins may be connected with certain climatic or soil conditions.

the results of presented biotechnological investigations clearly documented that R. kirilowii callus tissues are able to produce characteristic active compounds and could be in taken into consideration as a source of important pharmaceutical constituents.

the summary of pharmacological and clinical investigations on R. kirilowii ex-tracts will be shown in part II.

acknowledgements

the study was supported by grant of the Ministry of Science and high educa-tion, project no n405 025 32/1687.

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RhoDIoLa KIRILowII – StAn BADAń I PeRSPeKtyWy ZAStoSoWAnIA W lecZnIctWIecZęść I. uPRAWA W gRuncIe oRAZ W WARunKAch In vItRo oRAZ BADAnIA SKłADu cheMIcZnego KoRZenI I tKAneK KAluSoWych

AnnA KRAjeWSKA-PAtAn1*, MIRoSłAWA FuRMAnoWA2, MARIolA DRegeR1, AlInA MścISZ,1 SeBAStIAn MIelcAReK1, MAłgoRZAtA KAnIA1, WAlDeMAR BuchWAlD1, MAReK BARAnIAK1, AgnIeSZKA PIetRoSIuK2, MonIKA Zych2, MonIKA KARASIeWIcZ1, AnnA BogAcZ1, RADoSłAW KujAWSKI1, PRZeMySłAW M. MRoZIKIeWIcZ1

1 Instytut Roślin i Przetworów Zielarskich ul. libelta 2761-707 Poznań

2 Katedra i Zakład Biologii i Botaniki Farmaceutycznej Warszawski uniwersytet Medycznyul. Banacha 102-097 Warszawa

*autor, do którego należy kierować korespondencję: tel.: 48 61 6659540, faks: 48 61 6659551, e-mail: [email protected]

S t r e s z c z e n i e

Różeniec Kiryłowa (Rhodiola kirilowii (Regel) Maxim. (rodzina gruboszowatych Crassulaceae) jest rośliną stosowaną w tradycyjnej medycynie w Azji Wschodniej, głównie w chinach,

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jako środek zapobiegający niekorzystnym zmianom w organizmie człowieka na skutek hipoksji związanej z przebywaniem na dużych wysokościach (choroba wysokogórska). na podstawie własnych prac oraz szerokiego przeglądu piśmiennictwa autorzy przedstawiają obecny stan badań nad R. kirilowii w dziedzinie upraw w gruncie, badań fitochemicznych i badań biotechnologicznych (kultury kalusa, mikrorozmnażanie in vitro, produkcja na-sion somatycznych, kultury korzeni transformowanych). Dzieląc substancje chemiczne na grupy, w artykule przedstawiono zidentyfikowane składniki wyciągów z korzeni oraz tkanek kalusowych.

Słowa kluczowe: Rhodiola kirilowii, medycyna tradycyjna, hipoksja, uprawy w gruncie, kultury in vitro, mikrorozmnażanie, korzenie transformowane, nasiona somatyczne, badania fitochemiczne, salidrozyd, rozawiny