Biologia, Bratislava, 61/4: 463—467, 2006 Section Botany DOI: 10.2478/s11756-006-0077-x

Lithospermum officinale callus produces shikalkin

Kamahldin Haghbeen1*, Valiolah Mozaffarian2, Fatemeh Ghaffari1, Elahe Pourazeezi1, Mohammad Saraji3 & Morteza Daliri Joupari1

1The National Institute for Genetic Engineering and Biotechnology, Tehran, Iran 2Iran Research Institute of Forests and Range lands, Tehran 3Department of Chemistry, Isfahan University of Technology, Isfahan, Iran

Abstract: To study biosynthetic abilities of Lithospermum officinale, callus formation from young leaves and stems of the was induced on Linsmaier-Skoog medium supplemented with 2,4-D (10−6 M) and kinetin (10−5 M). Maintaining the calli on this medium resulted in polyphenolic compounds production. Their transfer onto White medium containing IAA (10−7 M) and kinetin (10−5 M) resulted in the production of a red naphthoquinonic pigment named shikalkin. Shikalkin production from callus cultures was suppressed on the White medium containing NAA instead of IAA. This observation indicates that both shikalkin and polyphenolic acids biosynthetic pathways exist in the L. officinale callus cells and a regulatory system counterbalances the ratio of shikalkin to polyphenolic acids. Key words: biosynthetic potential, callus, Lithospermum officinale, polyphenolic acids, shikalkin Abbreviations: NAA – 1-naphthaleneacetic acid; 2,4-D – 2,4-dichlorophenoxyacetic acid; IAA – 3-indoleacetic acid; 4CCA – 4-coumaroyl CoA; GPP – geranylpyrophosphate; LS – Linsmaier-Skoog; MS – Murashige-Skoog; PHBA – p- hydroxybenzoic acid; PPA – polyphenolic acids; RA – rosmarinic acid; THF, tetrahydrofuran.

Introduction ways share a common biosynthetic sequence, at least in the early steps. Hence, both routes can be triggered in Shikalkin refers to the mixture of shikonin and alkannin the L. erythrorhizon cell culture and a regulatory sys- (Khan et al., 1983, Fig. 1). These enantiomers are nat- tem balances the ratio of shikalkin to PPA (Gaisser urally produced in different ratios in the roots of some &Heide, 1996). Therefore, the presence or absence of of family alongside with a num- one of these two classes of compounds does not essen- ber of their corresponding esters (Papageorgiou et tially relate to the genetic potential of the plant cells al., 1999). Shikalkin is famous to plant biotechnologists in Boraginaceae family. It could depend on the condi- since Fujita et al. (1981a,b) introduced its successful tions governing the regulatory points somewhere in the large scale production plan based on the root suspen- phenylpropanoid pathway. To evaluate this idea, it was sion cell culture of the Lithospermum erythrorhizon in decided to search this common biosynthetic course in 1981. Fruitful in vitro cell culture of the L. erythrorhi- another member of the Boraginaceae family. This pa- zon root provided the plant scientists with a suitable per qualitatively supports this proposal through reveal- model to study phenolic secondary metabolites path- ing the results of a study on the cell culture of Lithos- ways in detail. Results of such studies have disclosed permum officinale, whose root is normally rich in PPA that the L. erythrorhizon callus is also able to produce (Kelley et al., 1975). In contrast to L. erythrorhizon, polyphenolic acids (PPA) like rosmarinic acid (RA) restricted to East Asia, L. officinale is a wide-spread (Mizukami et al., 1992) and lithospermic acid (Ya- and readily cultivated plant (Kelley et al., 1975). mamoto et al., 2000) not observed in the root of this naturally. Besides, it has been shown that the L. erythrorhizon yield of PPA production by the cell Material and methods culture can be enhanced, if the production of shikalkin derivatives is inhibited by light (Gaisser & Heide, Chemicals 1996). All chemicals used in this work were taken from the au- These results suggest that shikalkin and PPA path- thentic samples. Sephadex LH-20, naphthazarin and phy-

* Corresponding author: P.O.Box:14155-6343 Tehran, Iran, e-mail: [email protected], phone No: +98(21)4580372

c 2006 Institute of Botany, Slovak Academy of Sciences 464 K. Haghbeen et al.

HO OH OH CO2H

PHB

HO OH 4CCA

CO2H OH Shikimic acid CO-SCOA CO2H COOH PHB-geranyl PPO transferase GPP OH

OH Rosmarinic acid synthase

OH 3 -(4-Hydroxyphenyl)lactic acid

CO2H

O OH

COOH O HO

2-O-(4-Coumaroyl)-3-(4-hydroxyphenyl)lactic acid OH O RR'

OH O COOH HO O OH

OH O HO Rosmarinic acid (RA) Shikonin (R = OH, R' = H) Alkannin (R = H, R' = OH) Fig. 1. The biosynthetic pathways of shikalkin (shikonin and alkannin) and PPA like RA are two branches of phenylpropanoid route, which is separated from each other at 4CCA point. tohormones were purchased from SigmaTM Chemical and Induction of shikalkin production Biochemical Company. Calli of L. officinale on LS medium were transferred on White medium supplemented with IAA and kinetin. The Plant samples concentration of copper, as abiotic elicitor, in the applied L. officinale samples were collected from Bagh-shad in the −1 medium was raised to the level of 0.3 mg L mentioned for east of Tehran (Iran) at the end of June. Arnebia euchroma M9 medium (FUJITA et al., 1981) and the sugar content was samples were collected from Dena strict in the central Za- −1 reduced to 25 g L . All the experiments were carried out gross Mountain at 3200 to 3500 m altitudes. The plant speci- ◦ in the dark at 25 C. The most effective concentrations of mens were determined by the botanist of the scientific board −7 IAA and kinetin for pigment production were 10 Mand of the Iran Research Institute of Forests and Range Lands, −6 10 M, respectively. Dr V. Mozaffarian, and the head of the Tehran University Herbarium, Professor A. Ghahreman. UV-Vis studies on the constituents of the L. officinale and Seed germination and callus induction A. euchroma roots L. officinale seeds were collected, weighed and sterilized. Spectrophotometric measurements were carried out using Healthy seeds were differently germinated, however, with a Beckman model V-550, UV-Vis-NIR spectrophotometer. negative results. Therefore, the hard crusts of some seeds Roots of all samples were cleaned and dried at 37 ◦C for 48 h. were scratched and the seeds were put onto Murashige- The dried roots of A. euchroma were powdered. L. officinale Skoog (MS) hormone-free medium or MS medium supple- root is composed of two distinct parts. The outer part (skin) mented with 2,4-D (10−6 M) and kinetin (10−5 M). Both is dark brown colored and the inner part is cream colored. groups of seeds were germinated in the green-house at 25 ◦C, Both parts were dried separately, then, grinded to powder. 24h photo-period, under 10 000 lux light intensity in less On the basis of the solubility and stability tests (data than two weeks. Then the explants excised from roots, not shown), radical-free tetrahydrofuran (THF) was se- leaflets, and stems were transferred onto MS and Linsmaier- lected as the most suitable solvent for extracting secondary Skoog (LS) media and kept at 25 ◦C in the dark. The leaf metabolites of both dried calli and roots of these plants. The and stem explants produced calli on both MS and LS me- THF extract of A. euchroma was neatly divided into three dia supplemented with 2,4-D and kinetin. The most effective fractions with water, n-hexane and CHCl3. Water soluble concentrations of kinetin and 2,4-D for the L. officinale seed fraction was without red pigment. Both the skin and the germination and callus formation were 10−5 Mand10−6 M, core parts of the L. officinale root were also extracted by respectively. Sucrose (50 g L−1) was used as carbon source THF, then, partitioned by n-hexane and acetonitrile. in all the above mentioned experiments. The resulting calli Each of the collected fractions was chromatographed were sub-cultured every three weeks. on a Sephadex LH-20 column (2.5 cm diameter and 75 cm Lithospermum officinale callus produces shikalkin 465 height) using a mixture of water and methanol as the mo- bile phase. The ratio of H2O/MeOH was varying between 1/99 up to 25/75 to bring about neat separation of the con- stituents on the column. Each of the collected components was dried at room temperature in the dark. UV-Vis of each constituent in the corresponding solvent was recorded in a range of 200 to 700 nm. The stability of each component in basic and acidic media was also determined by UV-Vis spectra.

Mass spectroscopy experiments Chemical ionization mass spectroscopy experiments were carried out by a Trio 1000 mass spectrometer (Fisons Instru- Fig. 2. The spectra of the THF extract from the inner part (core) ments, Manchester, England) using methane as the reagent of the L. officinale root; 1 – in acidic milieu and, 2 – in basic gas. The ion source temperature was 150 ◦C, Electron energy milieu. Spectra 3 and 4 in the inset section belong to the THF extract of the outer part (skin) of the L. officinale root in acidic at 58eV, and the scan rate was 2 scans/sec. The tempera- and basic milieu, respectively. ture of the solid probe was programmed as follow: Initially 30 ◦C for 2 min, then increased to 140 ◦C (100 ◦Cmin−1,1 min hold) and subsequently 340 ◦C (300 ◦Cmin−1, 2 min hold). The recorded spectra were analyzed by the software provided by the company, Masslab version 1.3.

Results and discussion

It is well known that shikalkin derivatives are natu- rally produced in the roots of some Boraginaceae plants like L. erythrorhizon, A.euchroma and Alkanna tincto- ria (Papageorgiouet al., 1999; Tanaka et al., 1986; Xu-Qing & De-Wei, 1999). However, L. officinale is known for its PPA content (Kelley et al., 1975; De- Eknamkul & Ellis, 1984). Its root and leaves extracts Fig. 3. In contrast to the spectra shown in Fig. 2, naphtho- show antigonadotropic and antithyrotropic properties quinonic dyes usually have absorption bands above 460 nm. The spectra belong to 4 constituents separated from n-hexane and (Winterhoff et al., 1983). It was also shown that CHCl3 fractions of the THF extract of the A. euchroma root. the extracts, through inhibition of thyroid-stimulating- See Materials and methods for details. hormone, decrease the concentration of thyroxin and triidothyronine (Winterhoff et al., 1988; John et al., 1990). The medicinal and antioxidant activity of of the former group hardly exceed 460 nm, those of the L. officinale extract have also been ascribed to its PPA latter compounds extend to 700 nm with characteristic content like lithospermic acid and RA (Nahrstedt et pattern of naphthazarin derivatives (Van Der Vijver al., 1990; Chen & Ho, 1997). & Gerritsma, 1975; Agata et al., 1989; Tanaka et Although the roots of A. euchroma were dark-red al., 1989) (Figs 2,3). The UV-Vis spectra of the isolated colored, none of the roots of the collected L. officinale constituents from L. officinale root, both the skin and samples used in this work contained red or purple pig- core parts, did not show any peak beyond 460 nm even ment. The root was about 6 mm thick and consisted in basic milieu (Fig. 2). It suggests that the studied nat- of two distinct parts; skin and core. The skin part was ural L. officinale roots contained little naphthoquinonic dark-brown, while the inner part was colorless. THF ex- derivatives. tract of the inner part of the root was colorless, but its In order to examine biosynthetic potential of L. of- colorless water extract gradually developed an orange ficinale cells, its calli were obtained from leaflets and color. This colorful extract turned brown after heat- stems. However, the explants of roots as well as of ing (Kelley et al., 1975), which was still easily distin- young plants failed to form callus. The callus induced guishable from the characteristic color of the naphthaz- from leaves and stems showed best growth rates, up to arin derivatives of 1,4-naphthoquinone. Derivatives like 300% in 3 weeks, and friability when sub-cultured on shikalkin show strong red color in neutral and acidic so- LS medium supplemented with 2,4-D (10−6 M), kinetin lutions and produce blue color in a basic milieu (Chen (10−5 M), and sucrose (5%). For shikalkin production, et al., 1996). The skin or core section of L. officinale calli were transferred onto White medium (Fujita et root extracts produced mainly yellow to orange color al., 1981a). The concentration of copper, as an elicitor, in basic milieu, no green or blue color was observed. was increased to 0.3 g L−1 (Fujita et al., 1981b) and Spectrophotometric patterns of PPA and 1,4- sucrose was reduced to 2.5% to establish suitable car- naphthoquinone derivatives are clearly different (Wil- bon/nitrogen ratio for the secondary metabolites pro- liams & Fleming, 1980); while the absorption spectra duction (Srinivasan & Ryu, 1993). Calli were then 466 K. Haghbeen et al.

Fig. 4. The obtained L. officinale callus on (A) LS (2,4-D, 10−6 M), (B) White (NAA, 10−6 M), and (C) White (IAA, 10−7 M) media supplemented with kinetin (10−5 M).

tracted pigment from A. euchroma root resulted in the similar Rf (s) (Fig. 5). Both pigments were studied also by chemical ionization -mass spectroscopy which results in a softer ionization of the metabolites with strong enough “[MH]+, [M+Reagent gas]+”peaks(Maloney, 2004). The existence of shikalkin could be detected from + + 289 [MH] , 305 [M+CH5] peaks with intensities of 12 and 7, respectively (Fig. 6). Moreover, the advent of some known intermediate peaks produced from the de- struction of shikalkin derivatives at 274, 219, and 192 were in favor of the naphthoquinonic derivatives exis- tence (Shukla et al., 1971). These evidences indicate that L. officinale cells, besides their natural ability to produce PPA, have the potential to produce naphthoquinonic pigment. Detailed studies had already confirmed formation of Fig. 5. Pigment produced by L. officinale cells was gently washed PPA beside shikonin and some of its derivatives in from the surface of the callus by THF. After evaporation of the the root cell culture of L. erythrorhizon (Mizukami collected THF at room temperature in the dark, the pigment was et al., 1992, 1993). There are two precursors for fractioned by n-hexane and chromatographed on silica gel glass A. shikalkin formation, geranylpyrophosphate (GPP) and plate against the n-hexane fraction of the THF extract of the p euchroma root. A mixture of acetonitrile and MeOH (1/3) was -hydroxybenzoic acid (PHBA). GPP is made by con- used as mobile phase. densation of two mevalonic acids coming from iso- prenoid pathway, and PHBA is derived from phenyl- propanoid route (Fig. 1) (Khan et al., 1983; Pa- divided into two groups. One group was supplemented pageorgiou et al., 1999; Yazaki et al., 1997). On with kinetin and NAA and the other one with kinetin the other hand in RA biosynthesis as an example and IAA. Calli of the first group started to darken grad- of PPA, 2-O-(4-coumaroyl)-3-(4-hydroxyphenyl)lactic ually and excretion of brown colored metabolites into acid is known as a precursor (Matsuno et al., the medium was observed, especially after the second 2001). This intermediate, in turn, is formed by con- subculture. However, they showed no red or purple pig- densation of 4-coumaroyl CoA (4CCA) and 3-(4- ment production, even after one year of culture. In con- hydroxyphenyl)lactic acid (Fig. 1). The crucial point trast, one sample of the second group started to pro- is the 4CCA, which is the key intermediate for PHBA duce the red pigment already after the first subculture (Yamamoto et al., 2002). Results obtained in this work (Fig. 4). support the idea that both shikalkin and PPA biosyn- Although TLC experiment of the produced pig- thetic pathways have common steps up to 4CCA for- ment by L. officinale cells in comparison with the ex- mation. The conversion of 4CCA to PHBA seems to

Fig. 6. The chemical ionization-mass spectra of the samples introduced in Fig. 5. See Materials and methods for details of spectroscopy experiments. Lithospermum officinale callus produces shikalkin 467 be an important regulatory step in shikalkin biosynthe- MIZUKAMI,H.,TABIRA,Y.&ELLIS, B. 1993. Methyl jasmonate- sis. induced rosmarinic acid biosynthesis in Lithospermum ery- throrhizon 12: Invaluable studies have already revealed the key cell suspension cultures. Plant Cell Rep. 706– 709. function of phenylalanine lyase in phenylpropanoid MIZUKAMI,H.,OGAWA,T.,OHASHI,&H.ELLIS, B. 1992. In- pathway. However, it is not the unique control point of duction of rosmarinic acid biosynthesis in Lithospermum ery- this pathway, especially where it branches toward com- throrhizon cell suspension cultures by yeast extract. Plant 11: pounds such as PPA or shikalkin. It seems that factors Cell Rep. 480–483. 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