Plant Cell Reports (2000) 19 : 500503

Q Springer-Verlag 2000

J.L. Caruso 7 J. Callahan 7 C. DeChant K. Jayasimhulu 7 G.D. Winget

Carnosic acid in green callus and regenerated shoots of Rosmarinus officinalis

Received: 24 September / Revision received: 3 June 1999 / Accepted: 17 August

Abstract Rooted regenerated shoots obtained from leaf and nodal segments of Rosmarinus officinalis were grown on a basal nutrient medium for 9 weeks. The regenerants were shown by means of HPLC and mass spectrometry to contain carnosic acid, a diterpenoid with antioxidant and medicinal properties. Five-weekold nodular green callus also contained carnosic acid, whereas non-green, undifferentiated callus maintained in the dark did not. Key words Rosmarinus officinalis 7 Labiatae (Lamiaceae) 7 Tissue culture 7 Diterpene 7 Carnosic acid Abbreviations IAA: Indole-3-acetic acid 7 IBA: Indole-3-butyric acid 7 HPLC: High performance liquid chromatography 7 MS: Mass spectrometry 7 BAP: 6-Benzylaminopurine

order to increase the yield of desired products. What is not yet certain, however, is whether all cultured tissues or regenerated shoots will produce a desired product. We have used HPLC and MS to show and confirm, respectively, the presence of carnosic acid in both green differentiating callus and in shoots of regenerated intact rosemary plants.

Materials and methods

Plant material Mature specimens of rosemary (Rosmarinus officinalis) started from Standard Variety commercial seed (Richters, The Herb Specialists, Goodwood, Ontario, Canada) and grown at ambient temperature in the greenhouse were used in the initial extraction and purification of carnosic acid. These plants were also used as the source of material for tissue culture experiments. Tissue culture Shoot tips, measuring 3 cm in length, were dipped in 95% EtOH for 15 s, stirred in 10% diluted commercial bleach for 15 min, and rinsed three times in autoclaved distilled water. Leaf segments were trimmed to 5 mm in length and placed on media with their abaxial surface down. Stem sections that included a node 4 mm in length were placed upright in 60!15-mm petri dishes containing Murashige and Skoog (1962) nutrient medium supplemented with 3% sucrose (w/v) and either zeatin [6-(4-hydroxy-3-methylbut-2enylamino)purine], BAP, or thidiazuron [1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea], alone at 1 or 2 mg/l, or in combination with 0.1 mg/l IAA. Cultures were kept at ambient temperature (approx. 22 7C) under a 14-h photoperiod at a light intensity of 300 lux provided by cool-white lamps. Regenerated shoots were transferred to 150!25-mm tubes containing basal medium with 1.5% sucrose and IBA at 3 mg/l under the same growth conditions. Rooted regenerants were transferred to 200!25-mm tubes containing basal medium with 1.5% sucrose and kept under approximately 600 lux for the same photoperiod. Small amounts of callus initially grown on the growth regulator-containing media were also transferred to a basal medium containing 2 mg/l 2,4-D. These cultures were grown in the dark. Extraction One gram of dry leaves of mature plants grown from seed was ground for 4 min on ice in 12 ml reagent-grade acetone using a

Introduction
Carnosic acid is an important diterpenoid in rosemary. The chemical has utility as an antioxidant (Chen et al. 1992; Cuvelier et al. 1994; Haraguchi et al. 1995; Wenkert et al. 1965) and has medicinal properties (Paris et al. 1993; Offord et al. 1995). Smith (1996) suggested that plants arising from tissue culture can be a source of useful natural products. Tissue culture also has the potential to facilitate genetic manipulation in
Communicated by J.F. Finer J.L. Caruso 7 J. Callahan 7 C. DeChant 7 G.D. Winget (Y) Department of Biological Sciences, University of Cincinnati, P.O. Box 210006, Cincinnati, Ohio 45221-0006, USA e-mail: Doug.Winget@UC.EDU Fax: c513-556-5299 K. Jayasimhulu Department of Environmental Health, School of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA

501 polytron mill. One to several grams (dry weight) of regenerating shoot primordia from callus or rooted, regenerated shoots were treated similarly. The slurry was centrifuged (10 min, 27,000 g) at 4 7C. One milliliter of this supernatant was passed through a 0.45mm nylon filter and stored at 4 7C in a sample vial with a septum cap to be used in the HPLC analysis. Four milliliters of 1% acetic acid was added to the remaining supernatant and then mixed with n-hexane (1 : 1 volume). The latter fraction was collected for partitioning against saturated NaHCO3. The separated aqueous layer was adjusted to pH 2, an equal volume of CH2Cl2 was added, and the organic layer was collected. The volume was reduced in vacuo and brought to dryness under N2. Dried samples were stored at 30 7C in small vials. These dried samples were redissolved in 10 ml CH2Cl2 just prior to analysis by MS.

HPLC Following the protocol of Okamura et al. (1994), we carried out a HPLC analysis of prepared samples using a Ranin Rabbit HP with a 10-ml pump and a Gilson 112 fixed-wavelength detector at 254 nm. The HPLC was controlled and data were collected by the Dynamax program run on a MacIntosh SE. A Whatman Partisil 5C8 WCS analytical column (4.6!250 mm) was used at room temperature (227B1 7C). The carrier solvent was (60 : 40) acetonitrile:aqueous phosphoric acid (0.1% w/v) at a flow-rate of 1.5 ml/ min. Samples of 25 ml were injected manually. Some samples were run under identical conditions on a HPLC system with automatic injection and a Milton-Roy Spectro-monitor 3100 variablewavelength detector at 230 nm or 254 nm. Sample peaks were collected manually.

Mass spectrometric determination A Kratos MS-80 high performance mass spectrometer operating at a resolution of 7500 was used for analysis. Three microliters from a 10-ml solution of the dried samples (see above) was placed in a capillary sample holder which was sealed at one end, and the solvent (CH2Cl2) was evaporated by a gentle stream of N2. The capillary was then placed in the receptacle of a direct insertion probe through a vacuum lock. The temperature of the source was maintained at 150 7C and the beam current was held at 100 mA. The ionization energy was 70 eV. The magnet was scanned from 500 to 30 amu repetitively at 10 s/decade, and the data were collected and processed by a DS-55 data system, based on a Data General NOVA-4 computer.

produced roots when transferred to a medium containing IBA. The rooted specimens were transferred to a basal nutrient medium in test tubes. After 9 weeks regenerants attained an average height of 8 cm, at which time the shoots were analyzed for the presence of carnosic acid. Older shoots that had roots were transferred to soil and grown to maturity in a greenhouse. Callus grown on 2,4-D in the dark increased in size but was non-green and remained undifferentiated throughout the culture period. This callus was also assayed for carnosic acid. Using HPLC analysis, we readily identified carnosic acid as a major component in the acetone extracts from mature shoots of rosemary grown from seed (Fig. 1). A component with the same retention time (9.6 min) was present in extracts similarly prepared from green, differentiating callus tissues that were cultured in vitro. The eluent corresponding to this peak was collected, and the elemental composition obtained by high resolution mass spectrometric analysis was that of carnosic acid (C20H48O4). Furthermore, the mass spectra of the compound extracted from regenerants and mature plants (Fig. 2) agreed with the mass spectral data reported by others (Cuvelier et al. 1994). For additional confirmation, a partially purified extract was methylated by exposure to ethereal diazomethane and also subjected to high resolution MS analysis. After this treatment, there was a major component that had an elemental composition and mass spectrum corresponding to the methyl ester of carnosic acid. No carnosic acid was found in non-green, undifferentiated callus grown on 2,4-D in the dark. Surprisingly, carnosic acid was found in the roots of regenerated shoots.

Results and discussion

Leaf and node explants produced shoots on a nutrient medium supplemented with any of the cytokinins tested, either alone, or in combination with IAA. Zeatin at 1 mg/l and IAA at 0.1 mg/l, however, proved to be the best combination of growth regulators for the production of green callus, followed by the formation of shoots. No significant difference was observed in rate of callus and shoot formation between leaf and node explants. In both cases, a green callus with very small nodules was observed 56 weeks following the initial transfer of explants to culture media. Some of these differentiating green calli were extracted for carnosic acid, and other pieces were given an additional 6 weeks, which allowed for the development of shoots which averaged 3 cm in length. An average of five shoots emerged from each callus. All of the shoots

Fig. 1 HPLC separation of acetone-soluble material from dried commercial rosemary leaf. The peak indicated by the arrow had a retention time identical to that of purified carnosic acid and that fraction was collected for mass spectral analysis. Quantitatively similar amounts of carnosic acid were found in samples from seed-grown plants, green callus, and regenerated specimens

502 Fig. 2 Mass spectrum of purified carnosic acid crystals obtained from rosemary leaf extract. Mass spectral analyses obtained from extracts of regenerated specimens were compared to this standard for identification

Carnosic acid and carnosol have been reported to account for over 90% of the antioxidant activity in rosemary extract (Offord et al. 1995). Both carnosic acid and carnosol have been shown to be stronger antioxidants than butylated hydroxytoluene and butylated hydroxyanisole in assays testing inhibition of lipoxygenase activity (Chen et al. 1992; Cuvelier et al. 1994). These diterpenes of rosemary were also shown to inhibit superoxide anion production in the xanthine oxidase system (Haraguchi et al. 1995). Of perhaps greater significance, carnosic acid has been shown to inhibit HIV protease activity in vitro (Paris et al. 1993). We explored the possibility of using tissue culture techniques to increase the rate of shoot formation and hence carnosic acid production in rosemary. It appears that the production of rooted regenerants 8 cm in height (approximately 3 months old) does not significantly reduce the time to establish equivalent-sized plants when compared to taking cuttings from seedgrown plants. Many clones, however, of selected genetic lines can be efficiently produced from cultured leaf segments or nodes. Carnosic acid, a diterpenoid, has long been known to be a major constituent of the terpenoids of rosemary, a member of the mint family. Kleinig (1989) reviewed the evidence that plastids, especially chloroplasts, are necessary for the formation of diterpenoids. His conclusion is supported by our finding that only green tissues in culture contained carnosic acid. Yet roots from whole plants contained carnosic acid, which suggests that carnosic acid (or a precursor) might be transported in the plant.

In many cases, it would be useful to increase the rate of production of desirable phytochemicals. While tissue culture offers the possibility of controlling the rate of metabolic pathways, there is no certainty that all of the necessary pathways are constitutive. Although rosemary already has been regenerated through tissue culture (Chaturvedi et al. 1984), our study provides the first evidence that carnosic acid is present in green callus as well as regenerated shoots of this important herb. Our finding that carnosic acid is also present in rosemary roots raises an interesting question regarding its site of synthesis.
Acknowledgments The authors wish to thank Reginald Crutcher and Letitia Edwards for technical assistance. This work was supported in part by the Dieckmann Forestry Chair of the University of Cincinnati.

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