Biotechnology Letters 23: 77–82, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 77 Supercritical CO2 extraction of velutinol A from Mandevilla velutina (Apocynaceae) cultured cells and MALDI-TOF MS analysis M. Maraschin1,∗ , J.A. Sugui2 , K.V. Wood3 , C. Bonham3 , F.M. Lanças4 , P.S. Araujo1 , R.A. Yunes5 , R. Verpoorte6 & J.D. Fontana2 1 University Federal of Santa Catarina/CCA, Plant Morphogenesis/Biochemistry Laboratory, P.O. Box 476, 88.049-900, Florianopolis, Brazil 2 UFPR/Chemo-Biotechnology Biomass Lab., Curitiba, Brazil 3 Purdue University, Biochemistry Department, West Lafayette, USA 4 USP/IQSC/Chromatography Lab., Sao Carlos, Brazil 5 UFSC/Chemistry Department, Florianopolis, Brazil 6 LACDR/Division of Pharmacognosy, Leiden University, Leiden, The Netherlands ∗ Author for correspondence (Fax: 55-48-3342014; E-mail: m2@cca.ufsc.br) Received 24 August 2000; Revisions requested 27 September 2000; Revisions received 27 October 2000; Accepted 27 October 2000 Key words: MALDI-TOF MS, Mandevilla velutina, secondary metabolites, supercritical fluid extraction, velutinol A Abstract MALDI-TOF MS analysis of supercritical CO2 extracted samples obtained from Mandevilla velutina cell cultures allowed the detection of the anti-bradykinin pregnanic steroid, velutinol A, using low amount of sample (1 g lyophilized cells), with minimum analyte isolation. Introduction Much attention has been paid in obtaining bradykinin (BK) – Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg – antagonists, a nonapeptide released from plasma globulin by snake venom and also by trypsin, that participates in several physiological and pathological processes. After the demonstration of a BKpotentiating effect by Bothrops jararaca venom, there was an increasing interest in kinin action but its physiological and pathological role in many biological systems still remains unknown, due to the lack of a selective and competitive kinin antagonist (Calixto et al. 1991). Aqueous/alcoholic extracts and some pure pregnane (velutinol A, e.g., Figure 1) compounds isolated from tubers of the Brazilian plant Mandevilla velutina selectively antagonize, in a concentrationdependent manner, functional responses to BK and related kinins in several smooth muscle preparations (Calixto et al. 1995). Based on these findings, the compounds mentioned are of interest for develop- ment of new antiinflammatory medicines. These antiBK compounds are secondary metabolites, chemically characterized as pregnanic steroids. However, only small amounts are found in crude plant extracts [tuber, 0.001% to 0.0001%, w/w] (Calixto et al. 1989) and large-scale biomass production by conventional methods seems not be economically feasible. M. velutina cell cultures have been thought to be a suitable production system, because both callus (Calixto et al. 1989) and cell suspension cultures (Maraschin 1998) produce such pregnane compounds in higher amounts than the plant [0.0032%, w/w] and revealed an antiBK action about 31- to 79-fold greater than that obtained from crude tuber (Calixto et al. 1989). These metabolites still represent only minor constituents of the cell biomass, so that sensitive detection methods are needed for further studies aiming at increasing the in vitro production of the active compounds. Over the last years, the application of supercritical fluid extraction (SFE) has continuously grown in several areas, supported by the development of new 78 Fig. 1. Velutinol A (15R, 16R, 20S)14,16:15,20:16,21-triepoxy-15-16-seco-14β,17α-pregn-5-ene-3β,15-diol – a steroidal pregnane isolated from tubers and cell cultures of Mandevilla velutina (Apocynaceae). automated and low-cost systems. The characterization and analysis of food, drugs, pharmaceuticals, and natural products have been performed with interesting results (Overmeyer et al. 1999). For example, the analysis of polyprenols in Ginkgo biloba leaves by SCCO2 revealed the existence of a C120 isoprenolog, which was not detected by previous chromatographic methods (Huh et al. 1992). Similarly, progresses in mass spectrometry techniques and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have also been reported. The latter has proved suitable for the determination of molecular weight of biomolecules as proteins, peptides, oligonucleotides, oligosaccharides and synthetic polymers. The highly efficient TOF mass detection method coupled with the relatively gentle MALDI ionization method allow the routine analyses of biomolecules, using in some cases as little as femtomole amounts of material (Pfenninger et al. 1999). As depicted, this highly sensitive and fast technique might be especially important when one is interested in screening any plant secondary metabolite (taxol, e.g., Gimon et al. 1994) or new compounds in cell culture systems with pharmaceutical potential. This study was carried out in order to evaluate the feasibility of the application of SFE and MALDITOF MS techniques in detecting velutinol A in small amounts of Mandevilla velutina cultured cell biomass. Material and methods Cell cultures Callus cultures of Mandevilla velutina were initiated using nodal segments (6.5–8.0 mg), from a single 4-month-old platelet native to Cerrado ecosystem (Coromandel, Minas Gerais State/Brazil), on MS medium (Murashige & Skoog 1962) supplemented with 2 mg 2,4-dichlorophenoxyacetic acid l−1 , 2 mg 6-benzylaminopurine l−1 , and 3 mg 6furfurylaminopurine l−1 . From these cultures, 0.5 g of 21-day-old cells were subcultured in liquid medium to obtain cell suspension cultures as previously described (Maraschin 1998). Cell suspensions were maintained in 250-ml Erlenmeyer flasks under continuous light (1100 lux) and shaking (110 rpm), at 24 ± 1 ◦ C. Subculturing was performed every three weeks. Supercritical fluid extraction The following experiments were carried out after the collection of 15 g cells (fresh wt) from 21-day-old cell cultures (inoculum density = 3 g cells/50 ml culture medium) and centrifugation (10 000 × g/5 min). The cell biomass was lyophilized and stored at −20 ◦ C. The compound of interest was extracted from 1 g lyophilized cells, using a laboratory home-made device unit previously described (Coelho et al. 1997). Acetone was selected as SFE modifier of polarity as 10% (v/v) of the CO2 supercritical streaming. Fifty ml was introduced into a high-pressure vessel (500 ml) in the screw cap with a T-connection to permit introduction of CO2 (99.9% purity) and an inlet for the 79 Fig. 2. Partial, positive ion, MALDI-TOF mass spectra (m/z range = 200–600 daltons) of the F5 cell line (a) and F7 cell line (b) SFE-samples of M. velutina cultured cells. The peak resulting from velutinol A (m/z 362) is labeled with an asterisk (∗ ). admission of the pressurized modifier, at supercritical pressure, to the extraction cell (stainless steel, 20 cm × 0.8 cm i.d.). The molar ratio CO2 :acetone was conjugated to adjust the mix to supercritical conditions, known their respective critical temperature and critical pressure (72 ◦ C, 40 atm, 235.5 ◦ C and 47 atm). The solvent extractant (critical temperature ≥ 45.5 ◦ C and critical pressure ≥ 77 atm) was equilibrated in contact with the cell biomass for 10 min, after the extraction pressure was reached by using N2 (110 atm). The pressure used during extraction, according to a dynamic mode, was adjusted through a high-pressure valve and monitored by a manometer, with a flow rate of 2 ml min−1 and the extraction temperature (60 ◦ C) was controlled by an oven thermostat. As collection vessel a glass tube (20 cm × 1.5 cm i.d.) containing 10 ml acetone was used, with a flow restrictor (stainless steel, 0.1 mm i.d.) fixed in its screw cap, through which the fluid vapor pressure was relieved. By using a cryogenic trap, the collection vessel was cooled during extraction, and acetone was re-liquefied as it passed through the restrictor. Along the time course of 80 Fig. 3. Partial, positive ion, MALDI-TOF mass spectra (m/z range = 200–600 daltons) of the SFE-samples: (a) tuber [1 g – dry weight basis] and (b) non-producing cell line of M. velutina. Notice the absence of the peak resulting from velutinol A (m/z = 362) in the spectra. the extraction (∼80 min), ten samples were collected, followed by total evaporation of the solvent under reduced pressure and stored in sealed vials at −20 ◦ C until used. MALDI-TOF MS analysis The detection of velutinol A in the SFE-samples was performed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, at the Purdue University Campus-wide Mass Spectrometry Center, with a PerSeptive Biosystems (Framingham, MA) Voyager MALDI-TOF spectrometer. A nitrogen laser giving a 337 ηm output was used and the spectra were taken in the positive-ion mode, using an accelerating voltage of 28 kV. All the SFE-samples were previously resuspended in minimum volume of acetone (∼50 µl) and mixed (1:20) with the matrix α-cyano-4-hydroxycinnamic acid −10 mg ml−1 of H2 O/trifluoracetic acid/acetonitrile (4:1:5, by vol.). A 1-µl aliquot was applied to the sample target and allowed to dry before analysis. Results and discussion The spectra of the SFE-samples revealed the presence of velutinol A (C21 H30 O5 , MW = 362) in M. velutina cultured cell lines, despite the very low amount of cell biomass (1 g – dry weight basis) used (Figures 2a, b). Velutinol A and their glycoside-derivatives were not detected in plant tuber sample, 1 g (Figure 3a) 81 following the same SFE/MALDI-TOF MS protocol, proving the higher efficiency of the in vitro system in respect to conventional culture methods to produce the metabolites of interest. Similar results were found for velutinol A and its steroidal glycoside (MV 8612, C60 H94 O5 , MW = 1182) yielded in cell suspension cultures when compared with native tissue (tuber) by TLC/OD (Maraschin 1998). However, the signal corresponding to velutinol A was not observed in all the spectra and it may be related to variations of the concentration of velutinol A in the samples, indicating the existence of cell lines with different velutinol A content, and also the fact that the MALDI analysis is being carried near the threshold of detection for velutinol A (notice how weak the signal is in Figure 2b). Further MALDI-TOF MS analysis showed the occurrence of producing and non-producing cell lines (Figure 3b), indicating that this approach can be used as a strategy for the identification of higher producing cell lines. More recently, a patent covering velutinol A and its glycosyl-derivatives were issued in Canada (Canadian Patent Application 2.217088, September 1997) and a biotechnological process for production of this compound seems to be appropriate, since its obtainment through agricultural methods and chemical synthesis have failed so far (Calixto et al. 1989, Maraschin 1998). Additionally, several studies focusing on the activity, mechanism of action and structureactivity relationship of the aglycone and glycosyl form of velutinol A (Calixto et al. 1991, 1995) have been carried out. Further, these compounds may be of interest in better understanding the mechanism of BK antagonists. Results of the MALDI-TOF analysis of 3deoxyanthocyanidins and anthocyanins in crude extracts from Sorghum bicolor tissues showed sensitivities at 15 pmol µl−1 for 3-deoxyanthocyanidins and as low as 5 pmol µl−1 for pure samples of anthocyanidin [pelargonidin] and anthocyanin [malvin] (Sugui et al. 1998). These findings indicate that the potential MALDI-TOF MS has as a powerful analytical tool for identifying new compounds and/or monitoring secondary metabolism in plant or animal cells and tissues. In fact, various examples demonstrating the feasibility of qualitative/quantitative analysis by MALDI including in vivo and in vitro metabolism of drugs and secondary metabolites in real biological matrices have appeared in the literature. Several results are very exciting and highlight the advantages MALDI has over conventional methods. For example, the simultaneous detection and quantification of cyclosporin A (CsA) and its major metabolite AM1 in blood using matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI/TOF MS) has been performed, with data showing a good agreement with the HPLC results for both analytes (Muddiman et al. 1995). The findings of this study indicate the possibility of detecting secondary metabolites in low amounts of M. velutina cell biomass, by using SFE and MALDITOF MS. Little fragmentation, high sensitivity and tolerance to contamination are the major advantages of this method, allowing facile identification and quantification of metabolites produced in vitro with minimum analyte isolation. This approach would seem to be of interest in plant biotechnology programs, where MALDI could be used for early identification and selection of high-producing cell lines, reducing the duration of this usually time-consuming step. Furthermore, monitoring for the occurrence of secondary metabolite along the time course of the culture might also be feasible, due to the small sample size needed for the analysis. References Calixto JB (1995) Multiple mechanisms of bradykinin-induced contractions in rat and guinea pig smooth muscles in vitro. Eur. J. Pharmacol. 281: 279–288. Calixto JB, Silva AL, Reis MS, Costa RMBFL, Yunes JA, Cruz AB, Yunes RA (1989) Demonstration of anti-bradykinin compounds in callus cultures of Mandevilla velutina. Braz. J. Med. Biol. 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