Caruso J. Callahan C. DeChant,2000

8/13/2019 Caruso J. Callahan C. DeChant,2000

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Plant Cell Reports (2000) 19:500503 Q Springer-Verlag 2000

J.L. Caruso 7 J. Callahan 7 C. DeChantK. Jayasimhulu 7 G.D. Winget

Carnosic acid in green callus and regenerated shootsof Rosmarinus officinalis

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

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, USAe-mail: [email protected]: c513-556-5299

K. JayasimhuluDepartment of Environmental Health, School of Medicine,University of Cincinnati, Cincinnati, OH 45267, USA

Abstract Rooted regenerated shoots obtained fromleaf and nodal segments of Rosmarinus officinalis weregrown on a basal nutrient medium for 9 weeks. Theregenerants were shown by means of HPLC and massspectrometry to contain carnosic acid, a diterpenoidwith antioxidant and medicinal properties. Five-week-old nodular green callus also contained carnosic acid,whereas non-green, undifferentiated callus maintainedin the dark did not.

Key words Rosmarinus officinalis 7 Labiatae(Lamiaceae) 7 Tissue culture 7 Diterpene 7 Carnosicacid

Abbreviations IAA: Indole-3-acetic acid 7 IBA:Indole-3-butyric acid 7 HPLC: High performanceliquid chromatography 7 MS: Mass spectrometry 7BAP: 6-Benzylaminopurine

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 bea source of useful natural products. Tissue culture alsohas the potential to facilitate genetic manipulation in

order to increase the yield of desired products. What isnot yet certain, however, is whether all cultured tissuesor 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 greendifferentiating callus and in shoots of regeneratedintact rosemary plants.

Materials and methods

Plant material

Mature specimens of rosemary (Rosmarinus officinalis) startedfrom Standard Variety commercial seed (Richters, The HerbSpecialists, Goodwood, Ontario, Canada) and grown at ambient

temperature in the greenhouse were used in the initial extractionand purification of carnosic acid. These plants were also used asthe source of material for tissue culture experiments.

Tissue culture

Shoot tips, measuring 3 cm in length, were dipped in 95% EtOHfor 15 s, stirred in 10% diluted commercial bleach for 15 min, andrinsed three times in autoclaved distilled water. Leaf segmentswere trimmed to 5 mm in length and placed on media with theirabaxial surface down. Stem sections that included a node 4 mm inlength were placed upright in 60!15-mm petri dishes containingMurashige and Skoog (1962) nutrient medium supplemented with3% sucrose (w/v) and either zeatin [6-(4-hydroxy-3-methylbut-2-enylamino)purine], BAP, or thidiazuron [1-phenyl-3-(1,2,3-thia-diazol-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 of300 lux provided by cool-white lamps. Regenerated shoots weretransferred to 150!25-mm tubes containing basal medium with1.5% sucrose and IBA at 3 mg/l under the same growth condi-tions. Rooted regenerants were transferred to 200!25-mm tubescontaining basal medium with 1.5% sucrose and kept underapproximately 600 lux for the same photoperiod. Small amountsof callus initially grown on the growth regulator-containing mediawere 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 wasground for 4 min on ice in 12 ml reagent-grade acetone using a


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polytron mill. One to several grams (dry weight) of regeneratingshoot primordia from callus or rooted, regenerated shoots weretreated similarly. The slurry was centrifuged (10 min, 27,000g) at4 7C. One milliliter of this supernatant was passed through a 0.45-mm nylon filter and stored at 4 7C in a sample vial with a septumcap to be used in the HPLC analysis. Four milliliters of 1% aceticacid was added to the remaining supernatant and then mixed withn-hexane (1:1 volume). The latter fraction was collected for parti-

tioning against saturated NaHCO3. The separated aqueous layerwas adjusted to pH 2, an equal volume of CH2Cl2 was added, andthe organic layer was collected. The volume was reduced in vacuoand brought to dryness under N2. Dried samples were stored at30 7C in small vials. These dried samples were redissolved in10 ml CH2Cl2 just prior to analysis by MS.

HPLC

Following the protocol of Okamura et al. (1994), we carried out aHPLC analysis of prepared samples using a Ranin Rabbit HPwith a 10-ml pump and a Gilson 112 fixed-wavelength detector at254 nm. The HPLC was controlled and data were collected by theDynamax program run on a MacIntosh SE. A Whatman Partisil5C8 WCS analytical column (4.6!250 mm) was used at room

temperature (227B1 7C). The carrier solvent was (60:40) acetoni-trile:aqueous phosphoric acid (0.1% w/v) at a flow-rate of 1.5 ml/min. Samples of 25 ml were injected manually. Some sampleswere run under identical conditions on a HPLC system with auto-matic injection and a Milton-Roy Spectro-monitor 3100 variable-wavelength detector at 230 nm or 254 nm. Sample peaks werecollected manually.

Mass spectrometric determination

A Kratos MS-80 high performance mass spectrometer operatingat a resolution of 7500 was used for analysis. Three microlitersfrom a 10-ml solution of the dried samples (see above) was placedin a capillary sample holder which was sealed at one end, and thesolvent (CH2Cl2) was evaporated by a gentle stream of N2. Thecapillary was then placed in the receptacle of a direct insertionprobe through a vacuum lock. The temperature of the source wasmaintained at 150 7C and the beam current was held at 100 mA.The ionization energy was 70 eV. The magnet was scanned from500 to 30 amu repetitively at 10 s/decade, and the data werecollected and processed by a DS-55 data system, based on a DataGeneral NOVA-4 computer.

Results and discussion

Leaf and node explants produced shoots on a nutrientmedium supplemented with any of the cytokininstested, either alone, or in combination with IAA.

Zeatin at 1 mg/l and IAA at 0.1 mg/l, however, provedto be the best combination of growth regulators for theproduction of green callus, followed by the formationof shoots. No significant difference was observed inrate of callus and shoot formation between leaf andnode explants. In both cases, a green callus with verysmall nodules was observed 56 weeks following theinitial transfer of explants to culture media. Some ofthese differentiating green calli were extracted forcarnosic acid, and other pieces were given an additional6 weeks, which allowed for the development of shootswhich averaged 3 cm in length. An average of fiveshoots emerged from each callus. All of the shoots

Fig. 1 HPLC separation of acetone-soluble material from driedcommercial rosemary leaf. The peak indicated by the arrow had aretention time identical to that of purified carnosic acid and thatfraction was collected for mass spectral analysis. Quantitativelysimilar amounts of carnosic acid were found in samples fromseed-grown plants, green callus, and regenerated specimens

produced roots when transferred to a mediumcontaining IBA. The rooted specimens were trans-ferred to a basal nutrient medium in test tubes. After 9weeks regenerants attained an average height of 8 cm,at which time the shoots were analyzed for the presenceof carnosic acid. Older shoots that had roots were

transferred to soil and grown to maturity in a green-house.Callus grown on 2,4-D in the dark increased in size

but was non-green and remained undifferentiatedthroughout the culture period. This callus was alsoassayed for carnosic acid.

Using HPLC analysis, we readily identified carnosicacid as a major component in the acetone extracts frommature shoots of rosemary grown from seed (Fig. 1). Acomponent with the same retention time (9.6 min) waspresent 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 resolu-tion mass spectrometric analysis was that of carnosicacid (C20H48O4). Furthermore, the mass spectra of thecompound extracted from regenerants and matureplants (Fig. 2) agreed with the mass spectral datareported by others (Cuvelier et al. 1994). For additionalconfirmation, a partially purified extract was methy-lated by exposure to ethereal diazomethane and alsosubjected to high resolution MS analysis. After thistreatment, there was a major component that had anelemental composition and mass spectrum corre-sponding to the methyl ester of carnosic acid.

No carnosic acid was found in non-green, undiffer-entiated callus grown on 2,4-D in the dark. Surpris-ingly, carnosic acid was found in the roots of regener-ated shoots.


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Fig. 2 Mass spectrum ofpurified carnosic acid crystalsobtained from rosemary leafextract. Mass spectral analysesobtained from extracts ofregenerated specimens werecompared to this standard foridentification

Carnosic acid and carnosol have been reported toaccount for over 90% of the antioxidant activity inrosemary extract (Offord et al. 1995). Both carnosicacid and carnosol have been shown to be strongerantioxidants than butylated hydroxytoluene and buty-lated hydroxyanisole in assays testing inhibition oflipoxygenase activity (Chen et al. 1992; Cuvelier et al.1994). These diterpenes of rosemary were also shownto inhibit superoxide anion production in the xanthineoxidase system (Haraguchi et al. 1995). Of perhapsgreater significance, carnosic acid has been shown toinhibit HIV protease activity in vitro (Paris et al.1993).

We explored the possibility of using tissue culturetechniques to increase the rate of shoot formation andhence carnosic acid production in rosemary. It appearsthat the production of rooted regenerants 8 cm inheight (approximately 3 months old) does not signifi-cantly reduce the time to establish equivalent-sizedplants when compared to taking cuttings from seed-

grown plants. Many clones, however, of selectedgenetic lines can be efficiently produced from culturedleaf segments or nodes.

Carnosic acid, a diterpenoid, has long been knownto be a major constituent of the terpenoids of rosemary,a member of the mint family. Kleinig (1989) reviewedthe evidence that plastids, especially chloroplasts, arenecessary for the formation of diterpenoids. His conclu-sion is supported by our finding that only green tissuesin culture contained carnosic acid. Yet roots fromwhole plants contained carnosic acid, which suggeststhat carnosic acid (or a precursor) might be transportedin the plant.

In many cases, it would be useful to increase the rateof production of desirable phytochemicals. While tissueculture offers the possibility of controlling the rate ofmetabolic pathways, there is no certainty that all of thenecessary pathways are constitutive. Although rose-mary already has been regenerated through tissueculture (Chaturvedi et al. 1984), our study provides thefirst evidence that carnosic acid is present in greencallus as well as regenerated shoots of this importantherb. Our finding that carnosic acid is also present inrosemary roots raises an interesting question regardingits site of synthesis.

Acknowledgments The authors wish to thank Reginald Crutcherand Letitia Edwards for technical assistance. This work wassupported in part by the Dieckmann Forestry Chair of theUniversity of Cincinnati.

References

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Chen Q, Shi H, Ho C (1992) Effects of rosemary extracts andmajor constituents on lipid oxidation and soybean lipoxy-genase activity. J Am Oil Chem Soc 69:9991002

Cuvelier M, Berset C, Richard H (1994) Antioxidant constituentsin sage (Salvia officinalis). J Agric Food Chem 42:665669

Haraguchi H, Saito T, Okamura N, Yagi A (1995) Inhibition oflipid peroxidation and superoxide generation by diterpenoidsfrom Rosmarinus officinalis. Plant Med 61:333336

Kleinig H (1989) The role of plastids in isoprenoid biosynthesis.Annu Rev Plant Physiol Plant Mol Biol 40:3959

Murashige T, Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiol Plant15:473497


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Okamura N, Fujimoto Y, Kuwabara S, Yagi A (1994) High-performance liquid chromatographic determination ofcarnosic acid and carnosol in Rosmarinus officinalis and Salviaofficinalis. J Chromatogr A 679: 381386

Paris A, Strukelj B, Renko M, Turk V, Puki M, Umek A, KorantBD (1993) Inhibitory effect of carnosic acid on HIV-1protease in cell-free assays. J Nat Prod 56:14261430

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Caruso J. Callahan C. DeChant,2000

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