, Volume 223, Issue 2, pp 369–373

Production of rosmarinic acid and a new rosmarinic acid 3′-O-β-D-glucoside in suspension cultures of the hornwort Anthoceros agrestis Paton


  • Katharina Vogelsang
    • Institut für Pharmazeutische BiologiePhilipps-Universität Marburg
  • Bernd Schneider
    • Max-Planck-Institut für Chemische ÖkologieBeutenberg Campus
    • Institut für Pharmazeutische BiologiePhilipps-Universität Marburg
Original Article

DOI: 10.1007/s00425-005-0089-8

Cite this article as:
Vogelsang, K., Schneider, B. & Petersen, M. Planta (2006) 223: 369. doi:10.1007/s00425-005-0089-8


Cell suspension cultures of the hornwort Anthoceros agrestis Paton (Anthocerotaceae) were cultivated and characterized in CB-media containing 2 and 4% sucrose. The suspension cells accumulated rosmarinic acid up to 5.1% of the cell dry weight as well as caffeoyl-4′-hydroxyphenyllactate. Moreover, a more hydrophilic compound was detected which was isolated and identified as rosmarinic acid 3′-O-β-D-glucoside, a new rosmarinic acid derivative. This new rosmarinic acid derivative was found up to 1.0% of the cell dry weight in suspension cells of A. agrestis.


Anthoceros agrestis Paton (Anthocerotaceae)HornwortsRosmarinic acidRosmarinic acid 3′-O-β-glucosideSuspension cultures


Rosmarinic acid (Fig. 1) is a well-known natural product from higher plants, e.g. from species of the families Lamiaceae and Boraginaceae (Petersen and Simmonds 2003 for a review). Its occurrence in lower plants is less well known. Rosmarinic acid was first described as a constituent of hornworts in the species Anthoceros punctatus and Folioceros fuciformis (Takeda et al. 1990a, b). Rosmarinic acid was also extracted from Anthoceros agrestis, A. husnotii and A. laevigata (own data not published) and from species of the fern family Blechnaceae (Harborne 1966; Häusler et al. 1992). This finding raises the question whether the ability to synthesize and accumulate rosmarinic acid has been developed once or several times independently during evolution. Other constituents from hornworts are the lignan-like compounds anthocerotonic acid and megacerotonic acid (Takeda et al. 1990a, b). Cinnamic acid esters such as methyl caffeate and methyl 4-coumarate have been reported from A. laevis and A. punctatus (Mendez and Sanz-Cabanilles 1979) and further constituents, e.g. the alkaloid anthocerodiazonin, from A. agrestis (Trennheuser 1992; Trennheuser et al. 1994).
Fig. 1

Structural formulae of rosmarinic acid (1), rosmarinic acid 3′-O-β-D-glucoside (2) and caffeoyl-4′-hydroxyphenyllactic acid = isorinic acid (3)

The biosynthesis of rosmarinic acid has been fully elucidated in Coleus blumei (Lamiaceae; Petersen et al. 1993; Petersen 1997). In order to investigate whether the same biosynthetic pathway is followed in hornworts we have established suspension cultures of A. agrestis Paton (Anthocerotaceae). Several enzymes of rosmarinic acid biosynthesis have been found in cell-free protein extracts from Anthoceros cells, e.g. cinnamic acid 4-hydroxylase (Petersen 2003).

The sucrose content of the medium (2 or 4%) strongly influenced the amount of rosmarinic acid in suspension cells of A. agrestis (Petersen 2003). Furthermore, a more hydrophilic compound was detected which showed a similar UV spectrum as rosmarinic acid. We here describe the isolation and structure elucidation of this compound as rosmarinic acid 3′-O-β-D-glucoside (Fig. 1) as well as its accumulation characteristics in suspension cultures of A. agrestis.

Materials and methods

Suspension cultures of A. agrestis

Suspension cultures of A. agrestis were established from a callus culture kindly donated to us by Professor Binding, University of Kiel, Germany (Binding and Mordhorst 1991). This callus culture is routinely cultivated on hormone-free CB-medium (Petersen and Alfermann 1988) without NZ-amines and with 1% sucrose as carbohydrate source. Suspension cultures for characterization were cultivated in CB medium with 2% sucrose (CB2) or 4% sucrose (CB4) medium. Subcultivation was performed every 7 days by transfer of 4–5 g wet cells with a perforated spoon into 50 ml fresh medium in a 250 ml Erlenmeyer flask. Cultures were incubated on a rotary shaker (100 rpm) in continuous light (150 μmol photons m−2 s−1) at 25°C.

Characterization of A. agrestis suspension cultures

Suspension cultures inoculated into CB2 and CB4 medium were cultivated for two weeks with harvest of one flask every second day. The cells were separated from the culture medium by suction filtration. Conductivity and pH in the cell-free medium were measured with appropriate electrodes; the sugar concentration was determined refractometrically. The cells were weighed (fresh weight, FW), frozen at −20°C and lyophilized for 48 h for the determination of dry weight (DW). Lyophilized cells (50 mg) were suspended in 2 ml 70% aqueous ethanol and incubated for 20 min at 70°C in an ultrasonic bath with intermediary vigorous mixing. After centrifugation at 3,000 g for 10 min the supernatant was diluted 1:10 (v/v) with 50% aqueous methanol acidified with 0.01% H3PO4 (85%) and analyzed by HPLC as described below but with 50% aqueous methanol acidified with 0.01% H3PO4 (85%) as eluent and detection at 333 nm. The rosmarinic acid content was quantified with help of authentic rosmarinic acid (25 μM) as standard. The amount of rosmarinic acid 3′-O-β-D-glucoside (RA-Glc) was calculated on the basis of the molar extinction coefficient of rosmarinic acid.

Isolation of rosmarinic acid 3′-O-β-glucoside

Rosmarinic acid 3′-O-β-glucoside was isolated from 73 g FW 4-day-old suspension cells of A. agrestis cultivated in CB4-medium after freezing at −20°C and lyophilization (8.9 g DW). The cells were extracted three times with 89 ml 70% aqueous methanol by ultrasonication for 10 min. The solvent was evaporated to nearly dryness and the residue redissolved in 50% aqueous methanol. The solution was extracted several times with ethyl acetate in order to remove rosmarinic acid and other less polar compounds. The aqueous phases were concentrated to dryness in vacuum, redissolved in a small volume methanol and applied to silica gel TLC plates (Merck Kieselgel 60 F254 pre-activated by heating to 100°C for 10 min) and developed in ethyl acetate : H2O : methanol : formic acid acid 80:10:5:5 (by vol.). The newly isolated compound showed a Rf value of approximately 0.32 and displayed a bright blue fluorescence at 366 nm. The silica gel zones containing this compound were scraped off the plate and eluted with 50% aqueous methanol. Further purification was achieved by semi-preparative HPLC on a Hypersil ODS column (length 29 cm, diameter 8 mm) using 35% aqueous methanol acidified with 0.03% acetic acid as solvent at a flow of 3.4 ml min−1 and a detection wavelength of 333 nm. Only fractions containing the compound to be identified were collected. Before redissolving the compound for NMR analysis the HPLC solvent was removed by lyophilization.

Enzymatic hydrolysis of rosmarinic acid 3′-O-β-glucoside

The newly isolated compound was dissolved in 0.5 ml 0.1 M ammonium acetate buffer pH 6 and 10 mg Rhozyme HP150 (Pollock and Pool Ltd, Reading, UK) were added. After incubation at 30°C for 4 h the assay was acidified with 100 μl 6 N HCl and extracted with ethyl acetate (3×0.5 ml). The extracts were dried and redissolved for HPLC analysis in 100 μl 50% aqueous methanol acidified with 0.01% H3PO4. Analysis by HPLC was performed on a Hypersil ODS column with 50% aqueous methanol acidified with 0.01% H3PO4 as eluent as described (Petersen and Alfermann 1988). The compound before and after enzymatic cleavage was further chromatographed on a TLC plate (Merck Kieselgel 60 F254) with toluene : ethyl formiate : formic acid 5:4:1 (by vol.) together with reference compounds. Cleavage products showed the same Rf-values as caffeic acid and 3,4-dihydroxyphenyllactic acid.

In order to clarify the linkage of the glucose unit to rosmarinic acid, the newly isolated compound was incubated with α- and β-glucosidases (5 U amyloglucosidase from Aspergillus niger (Sigma), 5 U α-glucosidase type IV from yeast (Sigma), 2 U β-glucosidase from almonds (Fluka) in 0.5 ml 0.1 M morpholinoethanesulfonate buffer pH 5.0. After incubation for 24 h at 37°C the reactions were stopped with 50 μl 6 N HCl and extracted three times with 0.5 ml ethyl acetate. After evaporation of the ethyl acetate the residues were redissolved in 150 μl acidified aqueous 50% methanol and analyzed by HPLC as decribed above.

Spectroscopic methods

1H NMR, 1H−1H COSY, HMBC, HMQC, and ROESY spectra were recorded on a Bruker Avance DRX 500 NMR spectrometer using a cryogenic TXI probehead (Bruker BioSpin, Rheinstetten, Germany). 13C NMR data were obtained from HMBC and HMQC experiments. Methanol-d4 was used as a solvent and TMS as internal standard. ESI mass spectra was measured in the negative ion mode on a Micromass Quattro II tandem quadrupole mass spectrometer equipped with an electrospray (ESI) source (Mirormass, Manchester, UK). UV spectra were obtained in 50% aqueous methanol containing 0.01% H3PO4.

Rosmarinic acid 3′-O-β-D-glucopyranoside

Systematic name: 3-(3,4-Dihydroxyphenyl)-acrylic acid 1-carboxy-2-[4′-hydroxy-(3′-O-β-D-glucopyranosyl)-phenyl]-ethyl ester. UV (50% aqueous methanol, 0.01% H3PO4) λmax 331 nm; ESI-MS (70 eV): m/z 521 (rel. int. 100) [M], 359 (11) [M-Glc], daughter ion scan of m/z 521 (30 eV): m/z 359 [M-Glc], 197 [C9H7O4] (dihydroxyphenyllactic acid moiety), 179 [C9H9O5] (caffeic acid moiety); 1H NMR, aglucone: δ 7.50 (d, J=15.9 Hz, H-7), 7.16 (d, J=2.0 Hz, H-2′), 7.03 (d, J=2.0 Hz, H-2), 6.93 (dd, J=8.2, 2.0 Hz, H-6), 6.86 (dd, J=8.2, 2.0 Hz, H-6′), 6.76 (d, J=8.2 Hz, H-5), 6.75 (d, J=8.2 Hz, H-5′), 6.28 (d, J=15.9 Hz, H-8), 5.11 (dd, J=9.5, 3.3 Hz, H-8′), 3.14 (dd, J=14.3, 3.3 Hz, H-7′a), 2.99 (dd, J=14.3, 9.5 Hz, H-7′b), glucose moiety: δ 4.76 (d, J=7.2 Hz, H-1′′), 3.92 (dd, J=11.5, 1.9 Hz, H-6′′a), 3.72 (dd, J=11.5, 5.1 Hz, H-6′′b), 3.43–3.46 (m, H-2′′, H-3′′, H-4′′, H-5′′). 13C NMR, aglucone: δ 177.8 (C-9′), 169.0 (C-9), 150.2 (C-4), 147.5 (C-3), 147.4 (C-3′), 147.4 (C-4′), 147.2 (C-7), 131.5 (C-1′), 127.8 (C-1), 126.1 (C-6′), 123.5 (C-6), 120.1 (C-2′), 117.2 (C-5), 116.4 (C-5′), 116.1 (C-8), 115.4 (C-2), 78.0 (C-8′), 39.5 (C-7′), glucose moiety: δ 104.8 (C-1′′), 78.4 (C-3′′), 78.2 (C-5′′), 75.2 (C-2′′), 71.6 (C-4′′), 62.6 (C-6′′).


Detection and hydrolysis of rosmarinic acid 3′-O-β-D-glucoside

In order to elucidate the biosynthesis of rosmarinic acid in hornworts, we established suspension cultures of A. agrestis Paton (syn. A. crispulus; Anthocerotaceae) from an already established callus culture (Binding and Mordhorst 1991). These suspension cultures accumulated rosmarinic acid up to 5.1% of the cell dry weight (Petersen 2003). Caffeoyl-4′-hydroxyphenyllactic acid (= isorinic acid; Satake et al. 1999; Fig. 1), a biosynthetic precursor of rosmarinic acid (Petersen and Metzger 1993) was present in the ethanolic (70%) extracts from suspension cells of A. agrestis besides rosmarinic acid (data not shown). Moreover, a more hydrophilic compound displaying a UV/Vis-spectrum very similar to the one of rosmarinic acid was found. During HPLC separation on a Hypersil ODS column with 50% aqueous methanol acidified with 0.01% H3PO4 and diode array detection both compounds showed an absorption maximum at 331 nm.

Enzymatic cleavage of the newly detected compound with the crude enzyme preparation Rhozyme HP150 (Petersen and Alfermann 1988) yielded as phenolic constituents caffeic acid and 3,4-dihydroxyphenyllactic acid (shown by HPLC and TLC analysis) and hence the same phenolic compounds as the hydrolysis of rosmarinic acid. Cleavage of the newly isolated compound with β-glucosidase yielded the aglycone rosmarinic acid, whereas α-glucosidases could not release rosmarinic acid.

Structure elucidation of rosmarinic acid 3′-O-β-D-glucopyranoside

The newly detected compound was isolated from 70% methanolic extracts from A. agrestis suspension cultures by preparative TLC and preparative HPLC. The structure of rosmarinic acid 3′-O-β-D-glucopyranoside (Fig. 1) was elucidated by spectroscopic methods. 1H−1H COSY, HMBC, and HMQC measurements confirmed the structure of the aglucon and a β-glucosyl unit. Chemical shift differences of protons in the 3,4-dihydroxyphenyllactic acid moiety, especially of H-2′ (δ 7.16) in comparison with the corresponding signals of rosmarinic acid (δH-2′ 6.74) and a corresponding cross signal in the HMBC spectrum indicated the glucose being attached to one of the phenolic hydroxyl groups at C-3′ or C-4′ of this phenyl ring. Yet, C-3′ and C-4′ resonated at the same frequency (δ 147.4), preventing discrimination of both positions by means of the HMBC spectrum. Therefore, a ROESY experiment was used to establish the attachment of the glucose unit to the hydroxyl group at C-3′. A strong nuclear Overhauser effect was observed between the anomeric H-1′′ (δ 4.67) and H-2′, which is consistent only with the 3′-O-glucoside. Further, correlation peaks in the ROESY spectrum between H-1′′ and H-7′a, H-7′b, and H-8′ confirmed this finding. Electrospray MS data were in complete accordance with the suggested structure of rosmarinic acid 3′-O-β-D-glucopyranoside as well.

Suspension cultures of A. agrestis: growth and accumulation of rosmarinic acid and rosmarinic acid 3′-O-β-glucoside

Suspension cultures of A. agrestis were characterized for two weeks in order to get more knowledge about the time course of the accumulation of rosmarinic acid and rosmarinic acid 3′-O-β-D-glucoside (Figs. 1 and 2). For this purpose the suspension cells were inoculated into CB-media (Petersen and Alfermann 1988) with 2% (CB2) and 4% (CB4) sucrose. Cells and cell-free media were harvested every second day. Growth was determined by the measurement of fresh weight and dry weight. In CB2-medium the fresh weight per flask increased from day 4 on until day 12 when the fresh weight of the cells in CB2-medium started to decline again. In CB4-medium fresh weight accumulation carried on nearly linearly until the end of the observation period. Dry weight accumulation only increased until day 6 of the culture period in CB2-medium but still increased until the end of the observation period in CB4-medium. The pH of the cell-free medium, both CB2 and CB4, quickly rose to values above pH 8.0. The consumption of ions, measured by the conductivity of the cell-free medium, declined from about 4 mS/cm to around 2.4 mS/cm on day 10. Then the conductivity of the CB2-medium increased again indicating cell lysis of the A. agrestis cells whereas it further decreased in CB4-medium. The sugar content of the CB4-medium decreased continuously until the end of the observation period. In CB2-medium the sugar content reached minimal values on day 8 and increased again due to the release of sugars and/or other light-refracting compounds from the lysed cells. The rosmarinic acid content of the cells reached its maximum in CB2-cultured cells on day 8 with 5.1% of the cell dry weight. In CB4-medium, the levels were lower (3.4% of the cell dry weight) and were only reached on day 12. In contrast, the level of rosmarinic acid 3′-O-β-D-glucoside was higher in cells cultivated in CB4-medium. Overall, however, the levels of rosmarinic acid 3′-O-β-D-glucoside in this cell line were significantly lower than the rosmarinic acid contents. A maximum of 1.0% of the dry weight was already reached on day 4 in CB4-cultured cells; afterwards the amount decreased rapidly and might contribute to the accumulation of free rosmarinic acid. The highest amount of rosmarinic acid 3′-O-β-D-glucoside at 0.6% in cells from CB2-medium was detected on day 4 and decreased as well afterwards.
Fig. 2

Characterization of suspension cultures of Anthoceros agrestis cultivated in CB-media with 2% (CB2) and 4% (CB4) sucrose; DW dry weight, FW fresh weight, RA rosmarinic acid, RA-Glc rosmarinic acid 3′-O-β-D-glucoside. Sugar content, conductivity and pH were measured in the cell-free medium. Representative example of the Anthoceros cells during one culture period


Rosmarinic acid is a well-known constituent found in plant families all over the higher plant kingdom (Petersen and Simmonds 2003 and literature cited therein) and even in lower plants like hornworts (Takeda et al. 1990a,1990b; Petersen 2003). Less ubiquitous are glucosides of rosmarinic acid which have–to our knowledge–so far only been published from Helicteres isora (Sterculiaceae; Satake et al. 1999). In this plant species the glucose units are attached either to both para-situated hydroxyl-groups of rosmarinic acid or only to the 4-OH-group of the caffeic acid moiety. The rosmarinic acid derivative isolated by us from suspension cultures of the hornwort A. agrestis, however, has a glucose attached to the hydroxyl group at C3 of the dihydroxyphenyllactate moiety of rosmarinic acid and therefore is a new rosmarinic acid derivative: rosmarinic acid 3′-O-β-D-glucoside. Phytochemical investigations on Anthoceros species have so far not revealed this rosmarinic acid glucoside but a lignan derived from rosmarinic acid such as anthocerotonic acid (Takeda et al. 1999a, b) or an alkaloid, anthocerodiazonin (Trennheuser et al. 1994).

During a cultivation cycle of a suspension culture of A. agrestis rosmarinic acid 3′-O-β-D-glucoside reached its highest accumulation with 1.0% of the cell dry weight in medium with 4% sucrose earlier (day 4) than the maximum of rosmarinic acid accumulation (day 12) with 3.4%. Interestingly, in medium with 2% sucrose the rosmarinic acid accumulation was higher with 5.1% of the cell dry weight reached at day 8, but the glucoside was accumulated to a lower extent (0.6% on day 4). Rosmarinic acid 3′-O-β-glucoside might therefore serve as a precursor for free rosmarinic acid, but cannot fully contribute to its accumulation.


We thank Dr. Aleš Svatoš (Jena, Germany) for mass spectrometric analysis.

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© Springer-Verlag 2005