Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, PO Box 55, FIN University of Helsinki, Finland

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1 52 Phytochemical A. VERMA Analysis ET AL. Phytochem. Anal. 19: (2008) Published online 24 July 2007 in Wiley InterScience ( DOI: /pca.1015 Phytochemical Analysis Optimisation of Supercritical Fluid Extraction of Indole Alkaloids from Catharanthus roseus using Experimental Design Methodology Comparison with other Extraction Techniques ARVIND VERMA, KARI HARTONEN* and MARJA-LIISA RIEKKOLA Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, PO Box 55, FIN University of Helsinki, Finland Received 31 August 2006; Revised 4 June 2007; Accepted 4 June 2007 Abstract: Response surface modelling, using MODDE 6 software for Design of Experiments and Optimisation, was applied to optimise supercritical fluid extraction (SFE) conditions for the extraction of indole alkaloids from the dried leaves of Catharanthus roseus. The effects of pressure ( bar), temperature (40 80 C), modifier concentration ( vol%) and dynamic extraction time (20 60 min) on the yield of alkaloids were evaluated. The extracts were analysed by high-performance liquid chromatography and the analytes were identified using ion trap-electrospray ionisation mass spectrometry. The method was linear for alkaloid concentration in the range μg/ml. The limits of detection and quantification for catharanthine, vindoline, vinblastine and vincristine were 0.2, 0.15, 0.1 and 0.08 μg/ml and 2.7, 2.0, 1.3 and 1.1 μg/g, respectively. The dry weight content of major alkaloids in the plants were compared using different extraction methods, i.e. SFE, Soxhlet extraction, solid liquid extraction with sonication and hot water extraction at various temperatures. The extraction techniques were also compared in terms of reproducibility, selectivity and analyte recoveries. Relative standard deviations for the major alkaloids varied from 4.1 to 17.5% in different extraction methods. The best recoveries (100%) for catharanthine were obtained by SFE at 250 bar and 80 C using 6.6 vol% methanol as modifier for 40 min, for vindoline by Soxhlet extraction using dichloromethane in a reflux for 16 h, and for 3,4 - anhydrovinblastine by solid liquid extraction using a solution of 0.5 M sulphuric acid and methanol (3:1 v/v) in an ultrasonic bath for 3 h. Copyright 2007 John Wiley & Sons, Ltd. Keywords: Supercritical fluid extraction; indole alkaloids; Catharanthus roseus; experimental design; liquid chromatography; mass spectrometry. INTRODUCTION Catharanthus roseus (L.) G. Don is a well-known medicinal plant belonging to the family Apocynaceae. It is regarded as a rich source of pharmaceutically important terpenoid indole alkaloids. Vindoline, catharanthine and 3,4 -anhydrovinblastine are its major alkaloids (Naaranlahti et al., 1991; Sottomayor et al., 1998) as well as precursors in the biosynthetic pathways of vinblastine and vincristine. The two latter alkaloids are well-known anti-cancer drugs used in the treatment of acute leukaemia and Hodgkin s disease (Noble, 1990). 3,4 -Anhydrovinblastine has been claimed for use as anti-neoplastic agent and has shown excellent preliminary results in reducing tumours relating to human lung, cervical and colon cancers as well as to non-hodgkin s lymphoma. This compound is being * Correspondence to: K. Hartonen, Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, PO Box 55, FIN University of Helsinki, Finland. kari.hartonen@helsinki.fi Contract/grant sponsor: University of Helsinki; Contract/grant number: project developed as a new lead drug with improved therapeutic properties, demonstrating a significantly higher maximum tolerated dose and less toxicity than its parent and related compounds (Schmidt et al., 2003). Several extraction methods for vinblastine, vincristine and 3,4 -anhydrovinblastine have been developed in the past. Owing to the high economic value of these drugs, some of these methods have been patented and used for commercial production by research laboratories and pharmaceutical companies. Previously applied extraction methods involve solid liquid extraction, using ultrasonic bath with dilute acid or alcohol as solvents, followed by ph control and re-extraction with an organic solvent (Renaudin, 1984; Naaranlahti et al., 1987; Miura et al., 1987). These are long and tedious procedures employing a large quantity of toxic organic solvents, which is expensive, hazardous to use and generates waste that is costly to dispose of. Therefore, it would be desirable to develop new environmentally friendly methods that can be scaled up for commercial production. Supercritical fluid extraction (SFE) technology has been considered a good option for the extraction of natural products, particularly for food and pharmaceutical

2 SUPERCRITICAL FLUID EXTRACTION OF INDOLE ALKALOIDS FROM CATHARANTHUS ROSEUS 53 ingredients (Jarvis and Morgan, 1997; Hamburger et al., 2004). Supercritical carbon dioxide is the most common fluid used because of its physiological compatibility, non-toxicity, inflammability, inexpensiveness and availability. In addition, high selectivity, convenient critical parameters (T c = 31.1 C, P c = 73.8 bar) and environmental friendliness are other significant advantages of using carbon dioxide as supercritical fluid. SFE shows numerous advantages when compared with traditional Soxhlet and solid-liquid extraction of solid matrices such as plant material. Advantages include (i) faster and more efficient extractions, (ii) no residual solvent in the final product, which lowers the operating costs due to the reduction in post-processing and clean-up steps, and (iii) sensitive and relatively non-volatile compounds can be separated under thermally mild conditions without decomposition (Song et al., 1992). The non-polarity of carbon dioxide is often a drawback, but this limitation can be overcome through the addition of a small amount of a more polar solvent as modifier (Ollanketo et al., 2001), which undergoes dipole dipole interaction and hydrogen bonding with polar functional groups of the compounds of interest (Hamburger et al., 2004). This results in significant increases in the solubilities of polar compounds and in their extraction efficiencies. Several applications employing supercritical carbon dioxide with polar modifiers for the extraction of moderately polar to polar natural products have been published, including alkaloids (Heaton et al., 1993). Many alkaloids including thebaine, codeine and morphine from poppy (Janicot et al., 1990), coronaridine and voacangine from Tabernaemontana catherinensis (Pereira et al., 2004), cocaine from coca leaves (Brachet et al., 2000), purine alkaloids from Maté (Ilex paraguariensis) (Saldaña et al., 1999), colchicine and colchicoside from Colchicum autumnale (Ellington et al., 2003) and vindoline and vinblastine from Catharanthus roseus (Song et al., 1992, Choi et al., 2002) have been successfully extracted using SFE. In this work, we tested the applicability of the SFE process for the selective extraction of indole alkaloids from freeze-dried leaves of C. roseus using carbon dioxide as supercritical solvent. The compounds were analysed using HPLC-UV and identified by HPLC-ESI/MS. A quantitative determination of dry weight content (μg/g) and a comparative account of the relative recoveries (%) of the major alkaloids catharanthine, vindoline and 3,4 -anhydrovinblastine is presented using different extraction methods, i.e. SFE, Soxhlet extraction, solid liquid extraction with sonication and hot water extraction at various temperatures. Hot water extraction could be an environmentally friendly method for the extraction of plant secondary metabolites such as glycosides and flavonoids. Kim et al. (2004) used a hot water extraction method to extract secologanin, a monoterpenoid glycoside, which is also the precursor in the biosynthesis of Catharanthus alkaloids. Hot water was found to be a more efficient technique than conventional organic acid extraction to extract iridoid glycosides such as catapol and aucubin from Veronica lonifolia leaves (Suomi et al., 2000). Bergeron et al. (2005) extracted flavonoid glycosides and amino acids with accelerated solvent extraction (ASE) at 85 C using water and hot water extraction techniques. In order to compare the extraction efficiencies of Catharanthus alkaloids we used hot water extraction at different temperatures, as an alternative method. The aims of the present study were (i) to assess the suitability of supercritical carbon dioxide for the extraction of Catharanthus alkaloids, (ii) to optimise the variables that affect the extraction, such as pressure, temperature, time of extraction and the amount of modifier, using factorial design experiments and (iii) to compare the results with those obtained by other extraction methods. EXPERIMENTAL Reagents and chemicals Methanol (VWR International AB, Stockholm, Sweden), acetonitrile (far UV) and dichloromethane (Lab-Scan, Analytical Sciences, Ireland) were of HPLC grade. Methyl tert-butyl ether was from Rathburn Chemicals (Walkerburg, Scotland). Ammonium acetate was supplied by Merck (Darmstadt, Germany) and ammonia (25%), hydrochloric acid (min. 32%) and sulphuric acid (95 97%) from Riedel-de Haën (Seelze, Germany). The standards vindoline and catharanthine were purchased from LKT Laboratories Inc. (St Paul, MN, USA). Vinblastine and vincristine were from Alexis Biochemicals Corporation (San Diego, CA, USA). The internal standard, ajmalicine hydrochloride was from Roth (Karlsruhe, Germany). Water was obtained from the Milli-Q plus purification system (Millipore, Molsheim, France). Plant material A new variety of Catharanthus roseus, Petrus (Grant of Community Plant Variety Rights, decision no. EU ), developed at the University of Helsinki, was used for the extraction of alkaloids. The plants were grown in the greenhouse from February to June under the following conditions: temperature 24 C, humidity 60%, light intensity 4000 lx with a photoperiod of 16 h/day. After a growing period of 6 weeks, a total of 27 seedlings were selected and transferred into pots. Leaves were collected from adult plants, freeze-dried and crushed to provide a homogenised sample.

3 54 A. VERMA ET AL. SFE instrumentation Supercritical fluid extractions were performed with an automated ISCO SFX 3560 (Lincoln, NE, USA) instrument using 6 ml extraction vessels. The extraction vessels were filled with 100 mg of dried plant samples mixed with anhydrous sodium sulphate (J.T. Baker, Deventer, Holland). The extracted analytes were collected into 10 ml of methanol. The internal standard was added to the collection vials immediately after extraction. The collection temperature was +5 C. The carbon dioxide for extraction ( %, SFE grade) and for cooling (99.7%), was purchased from Oy AGA Ab (Espoo, Finland). The SFE instrument was equipped with a 260 ml syringe pump for the addition of carbon dioxide at a flow-rate of 1.5 ml/min and a manually controlled Jasco (Tokyo, Japan) PU-980 HPLC pump for addition of the modifier (methanol) at flow-rates of ml/min ( %). The restrictor temperature was set at 60 C in all extractions. HPLC analysis Each extract was evaporated to dryness under nitrogen and the residue was dissolved in 1.0 ml of methanol. Samples were filtered through a non-sterile 13 mm Millipore Millex syringe filter unit (0.45 μm membrane) and analysed with an Agilent (Santa Clara, CA, USA) 1050 HPLC system equipped with a UV detector set at 214 nm. Chromatographic separations were carried out on a Phenomenex (Torrance, CA, USA) Gemini C 18 column ( mm i.d.) packed with 5 μm particles having pore size 100 Å. The mobile phase contained 10 mm ammonium acetate buffer (ph 5.0), acetonitrile and methanol in proportions of 65:20:15 changing to 30:40:30 during 30 min linear gradient at a flow rate of 0.3 ml/min. The total analysis time was 36 min but all major alkaloids were eluted within 21 min. The injections (5 μl) were made by an autosampler with an injection needle. The data were collected and analysed using a Hewlett-Packard computing system (Agilent ChemStation for LC, Rev. A.09.01). HPLC-MS analysis The major alkaloids in the plant extract were identified with a Hewlett-Packard 1100 HPLC system coupled to an Esquire-LC ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany) using an electrospray ionisation (ESI) interface in the positive ionisation mode. The chromatographic conditions were the same as described above for HPLC. The source temperature was set at 365 C and the source voltage was constant at 3.5 kv. Nitrogen gas was used as sheath and nebuliser gas at 9.0 L/min and 40.0 psi. The ion trap was scanned from 100 to 900 Da. Software for the design of SFE experiments Response surface modelling, using MODDE 6 software for Design of Experiments and Optimisation (UMETRICS AB, SE Umeå, Sweden) was applied to optimise the SFE conditions. A full factorial design with four factors, temperature, pressure, modifier flow-rate and dynamic extraction time was created at two levels, comprising all the possible combinations of the factor levels. Initially 19 experiments (N) were designed by the software for four factors (p) at two levels (N = 2 p + 3 centre points). All optimisation experiments were randomly performed without replication. The measured response was calculated as the dry weight content (μg/g) from the peak areas of catharanthine and vindoline. Traditional extraction methods Solid liquid extraction. Plant samples (100 mg) were extracted with 10 ml solution of 0.5 M sulphuric acid and methanol (3:1 v/v, ph 1.4) in an ultrasonic bath for 3 h. The extracts were filtered through Schleicher & Schuell (Micro Science, Dassel, Germany) filter papers (90 mm) on a Büchner funnel, and the residue was re-extracted with another 10 ml of the same solvent for a further period of 1 h. The internal standard was added to the combined supernatant, which was filtered, made alkaline (ph 9.5) with 2 ml of 25% ammonia and then extracted with 2 10 ml of methyl tert-butyl ether. Ether fraction was separated and evaporated to dryness. The dry residue was dissolved in 1 ml of methanol, filtered (0.45 μm membrane), and a 5 μl aliquot was injected into the HPLC. Soxhlet extraction. Soxhlet extraction was performed by extracting 100 mg of the plant sample with 70 ml of methanol (boiling point 65.5 C) and dichloromethane (boiling point 40.5 C) separately, in a reflux for 16 h. The internal standard was added immediately after extraction. The extracts were evaporated to dryness and the residues were dissolved in 1 ml of methanol, filtered (0.45 μm membrane), and 5 μl aliquots were injected into the HPLC. Hot water extraction at 50, 70 and 90 C. Plant samples (100 mg) were extracted with 10 ml of water in hot water bath with constant shaking for 3 h at 50, 70 and 90 C separately. The extracts were filtered through Whatman no. 40 filter paper on a Büchner funnel and the residue was re-extracted with another 10 ml of water for 1 h. The internal standard was added to the

4 SUPERCRITICAL FLUID EXTRACTION OF INDOLE ALKALOIDS FROM CATHARANTHUS ROSEUS 55 Table 1 Dry weight content (μg/g) and RSD (%) of major alkaloids in different extraction methods Dry weight content of major alkaloids in μg/g (RSD %) Method of extraction CTR VDL AVLB SFE (6.5) (7.1) 77.8 (7.2) Sox M (4.1) Sox D (11.6) (11.4) 29.4 (14.0) SLE (15.9) (13.3) (14.5) HWE (6.4) (7.5) 12.4 (6.3) HWE (6.3) (10.8) 12.9 (13.0) HWE (11.5) 8.2 (11.9) 4.5 (17.5) For SFE n = 6; for other methods n = 3. CTR = catharanthine; VDL = vindoline; AVLB = 3,4 -anhydrovinblastine; SFE = supercritical fluid extraction; Sox M = Soxhlet extraction using methanol; Sox D = Soxhlet extraction using dichloromethane; SLE = solid liquid extraction; HWE = hot water extraction at 50, 70 and 90 C. combined supernatant, which was filtered, made alkaline (ph 9.5) with 2 ml of 25% ammonia and then extracted with methyl tert-butyl ether (2 10 ml). The ether fraction was separated and evaporated to dryness. The dry residue was dissolved in 1 ml of methanol, filtered (0.45 μm membrane), and a 5 μl aliquot was injected into the HPLC. RESULTS AND DISCUSSION HPLC method development The C. roseus plant extract is a complex mixture of several alkaloids with a wide range of polarities (Fig. 1) and different pk a values. The selectivity is highly affected by mobile phase ph because of the substantial differences between the pk a values of the alkaloids. The chromatographic profile and the total analysis time can be altered by making slight changes in the organic contents and the ph of the mobile phase (Theodoridis et al., 1997). Several HPLC methods have been developed in the past for the analysis of indole alkaloids of C. roseus using solvent systems having buffers including ammonium acetate (Naaranlahti et al., 1987; Chu et al., 1997), triethylamine (Kohl et al., 1983) and sodium or ammonium hydrogen phosphate (Renaudin, 1984; Miura et al., 1987). Although phosphate buffer is more commonly used for the analysis of basic alkaloids, ammonium acetate has an advantage of being compatible with HPLC-MS owing to its volatility. The addition of ammonium acetate to the mobile phase alters the ionic strength, stabilises the ph and results in improved peak shape and selectivity (Theodoridis et al., 1997). The described method was developed using 10 mm ammonium acetate (ph 5.0), methanol and acetonitrile as mobile phase, which resulted in a good chromatographic separation of standard alkaloids within 20 min by gradient elution [Fig. 2(A)]. A higher (20 mm) or lower (5 mm) concentration of ammonium acetate did not result in improved peak shape or better resolution. Hence, the buffer concentration (within the studied range) does not affect the chromatographic profile. Catharanthine, vindoline, vinblastine, vincristine, leurosine and 3,4 -anhydrovinblastine were separated by varying the ph of the mobile phase. Vinblastine always co-eluted with an unknown compound at phs 4.3, 5.0 and 5.6 [Fig. 2(B)], making it difficult to calculate its dry weight precisely. On increasing the ph of the mobile phase to 6.8, the separation of vinblastine and vincristine was achieved but leurocine co-eluted with another compound (Fig. 3). The repeatability of retention times at ph 5.0 (within the buffer range) was better than that at ph 6.8 (outside the buffer range). The relative standard deviation (RSD) varied from 0.1 to 0.4% at ph 5.0 and from 4.7 to 5.3% at ph 6.8. Increasing the ph of the mobile phase also resulted in an increase in the retention of the major alkaloids on the column, and the total elution time increased from 21 min (ph 5.0) to 31 min (ph 6.8). However, catharanthine, vindoline and 3,4 -anhydrovinblastine were well separated at ph 5.0 and their dry weight contents were determined quantitatively (Table 1). Serpentine was not observed in the plant extracts, whereas peaks of tabersonine and vindolinine were observed in the HPLC chromatograms [Figs 2(B) and 3]. Since standards for these alkaloids were not available, their dry weight contents could not be determined. In most of the earlier HPLC methods, detection wavelengths of 298, 280 or 254 nm have been used (Naaranlahti et al., 1987; Song et al., 1992; Volkov and Grodnitskaya, 1994, Chu et al., 1997). Uniyal et al. (2001) observed that catharanthine, vindoline, vinblastine and vincristine showed greater absorption at 220 nm as compared with that at 298, 280 or 254 nm. We used the detection wavelength of 214 nm because vindoline and other dimeric alkaloids absorb slightly better at this wavelength than at 220 nm (Verma A, Laakso I; unpublished results). A slight drift in the

5 56 A. VERMA ET AL. Figure 1 Chemical structures of Catharanthus alkaloids. The numbers 1 7 represent the peak numbers in Figs 2 and 3.

6 SUPERCRITICAL FLUID EXTRACTION OF INDOLE ALKALOIDS FROM CATHARANTHUS ROSEUS 57 Figure 2 HPLC chromatograms of (A) standard compounds and (B) leaf extract of C. roseus obtained by SFE (mobile phase consisted of 10 mm ammonium acetate buffer at ph = 5.0). Key to peak identity: 1 = catharanthine (R t = 7.6 min); 2 = ajmalicine (internal standard; R t = 12.4 min); 5 = vindoline (R t = 19.1 min); 6 = leurocine (R t = 20.1); 7 = 3,4 -anhydrovinblastine (R t = 21.0); and 8 = unknown compound (R t = 16.8 min) co-eluting with vinblastine (R t = 17.0); x = tabersonine/vindolinine (R t = 2.8/3.3). (For analytical protocols see the Experimental section.) baseline was observed at 214 nm; however, it did not affect the integration of the peaks. Quantitation Quantitation of the alkaloids was based on the internal standard method. Ajmalicine was used as an internal standard to quantify the major alkaloids in the leaves. The radioimmunoassay for ajmalicine and serpentine by Arens et al. (1978) showed that ajmalicine and serpentine are primarily found in the Catharanthus root system, whilst the green parts of the plant are free of ajmalicine and contain only small amounts of serpentine. The HPLC method developed by Naaranlahti et al. (1987) for the analysis of Catharanthus alkaloids also showed that ajmalicine is present only in root samples. In more recent works by Verpoorte et al. (2002) and Van der Heijden et al. (2004), the presence of ajmalicine in the green parts of the plant was discussed. In the present work, the leaf extracts obtained by SFE, solid liquid extraction, Soxhlet and hot water extraction methods were analysed without the addition of the internal standard ajmalicine (blanks). Since we could not detect the presence of ajmalicine in the blanks, this compound was chosen as the internal standard for the quantification of major leaf alkaloids. The HPLC-MS spectra of the leaf sample did not show

7 58 A. VERMA ET AL. Figure 3 HPLC chromatogram of leaf extract of C. roseus obtained by solid liquid extraction (mobile phase consisted of 10 mm ammonium acetate buffer at ph = 6.8). Key to peak identity: 1 = catharanthine (R t = 19.9 min); 2 = ajmalicine (internal standard; R t = min); 3 = vincristine (R t = 23.0 min); 4 = vinblastine (R t = 25.7); 5 = vindoline (R t = 21.8); and 7 = 3,4 -anhydrovinblastine (R t = 30.7); x = tabersonine/vindolinine (R t = 7.3/9.5). (For analytical protocols see the Experimental section.) Figure 4 Comparison of relative recoveries of the three major alkaloids of C. roseus (the best recovery obtained is represented as 100%). For SFE n = 6, and for all other extraction methods n = 3. SFE = supercritical fluid extraction; Sox M = Soxhlet extraction using methanol; Sox D = Soxhlet extraction using dichloromethane; SLE = solid liquid extraction using ultrasonic bath; and HWE = hot water extraction at 50, 70 and 90 C. CTR = catharanthine; VDL = vindoline; and AVLB = 3,4 -anhydrovinblastine. the presence of the molecular ion (M + H) + of ajmalicine at m/z 353. In all of the extraction methods the internal standard was added after the extraction in order to avoid its possible thermal degradation owing to high temperatures and long residence times of extraction. Ten-point calibration graphs (n = 3) were created for each of the four standards, namely catharanthine, vindoline, vinblastine and vincristine, for the range μg/ml by plotting the peak area ratios of analyte and internal standard vs the amounts of analytes. Linear regression analysis was used to calculate the calibration curve parameters. Good linearity was observed (r in all instances) for the above range. The limits of detection (LOD) were calculated from the lowest concentration of calibration standards for each compound based on three times the noise level. For catharanthine, vindoline, vinblastine and vincristine, the LOD were found to be 0.2, 0.15, 0.1 and 0.08 μg/ml, respectively. The noise level in the sample run was only slightly higher (four times) than that in the standard run. The limits of quantification (LOQ) were calculated to be 2.7, 2.0, 1.3 and 1.1 μg/g, respectively. Since the standard for 3,4 - anhydrovinblastine was not available, it was quantified in the plant sample using the calibration parameters of vinblastine.

8 SUPERCRITICAL FLUID EXTRACTION OF INDOLE ALKALOIDS FROM CATHARANTHUS ROSEUS 59 Table 2 Factor levels and design matrix (2 4 factorial design) for SFE Dry wt content (μg/g) Experiment Experiment Run P T Modifier flow Time No. name order (bar) ( C) (ml/min) (min) CTR VDL 1 N N N N N N N N N N N N N N N N N N N C C C C C C C C C Nineteen initial runs (N1 N19) for four factors (p) at two levels (N = 2 p + 3 centre points) and nine complimentary runs (C20 C28; two runs for each factor + one centre point) were designed by the software. Response is shown as dry weight content (μg/g) for catharanthine (CTR) and vindoline (VDL). HPLC-MS analysis Soft ionisation techniques such as atmospheric pressure chemical ionisation (APCI) or electrospray ionisation (ESI) have often been used for the characterisation of plant secondary metabolites because the MS so obtained are dominated by respective protonated molecules in high yields (Chu et al., 1997). The chromatographic profile of C. roseus is very complex due to the presence of a large number of different alkaloids, some of which co-elute with each other. Therefore, the major peaks in the leaf extracts were identified using HPLC-ESI/MS). For monomeric alkaloids, such as catharanthine and vindoline, only monoprotonated ions [M + H] + were observed, whilst for dimeric alkaloids, such as vinblastine, vincristine, leurocine and 3,4 -anhydrovinblastine, both mono- and diprotonated [M + 2H] 2+ ions were detected at their corresponding m/z values. Both tabersonine and vindolinine were identified by their protonated molecular ions (M + H) + at an m/z value of Optimisation of SFE conditions Response surface regression was used to obtain an optimum set of conditions. With 19 initial experiments (Table 2, Expt. N1 N19) no clear maximum or minimum response could be found within the design factor ranges used. Therefore, a complement design for all the four factors was made to allow the estimation of quadratic effects, evaluate how the factors influenced the response and to optimise or find a region of operability. This further proposed two runs per factor plus an additional centre point (Table 2, Expt. C20 C28). In this way, non-linear responses could be revealed and a clear optimum was found for each parameter. The runs were carried out in random order, and the response surface plots were drawn for each parameter. Catharanthine and vindoline were extracted in all 19 initial experiments (N1 N19) as well in the complementary experiments (C20 C28), whereas the peaks for the dimeric alkaloids vinblastine, vincristine, leurocine and 3,4 -anhydrovinblastine were present in only some of

9 60 A. VERMA ET AL. Figure 5 Response surface plots for (A) catharanthine and (B) vindoline showing their dry weight content (μg/g) as a function of temperature ( C) and pressure (bar); the other two factors (dynamic extraction time and modifier flow) were kept constant at their middle values. Figure 6 Response surface plots for catharanthine showing its dry weight content (μg/g), (A) as a function of temperature ( C) and dynamic extraction time (min), the other two factors (modifier flow and pressure) being kept constant at their middle values; (B) as a function of pressure (bar) and dynamic extraction time (min), the other two factors (modifier flow and temperature) being kept constant at their middle values. the initial and complementary runs. The response (dry weight content) could only be determined precisely for catharanthine and vindoline. The response surface plots were created for individual compounds as three-dimensional plots by presenting the response as a function of two factors and keeping the other two constant at their centre values. Six separate plots were made for both catharanthine and vindoline for all four parameters. The optimum set of conditions was determined from Figs 5 7. Other plots showing similar response surfaces (trends) are not presented. The effect of pressure and temperature on the dry weight content of catharanthine and vindoline clearly showed that high temperature (80 C) favours the extraction recoveries of both alkaloids. The recovery of catharanthine was highest at the lowest pressure

10 SUPERCRITICAL FLUID EXTRACTION OF INDOLE ALKALOIDS FROM CATHARANTHUS ROSEUS 61 Figure 7 (A) Response surface plot for catharanthine showing its dry weight content (μg/g) as a function of pressure (bar) and modifier flow (ml/min), the other two factors (temperature and dynamic extraction time) being kept constant at their middle values. (B) Response surface plot for vindoline showing its dry weight content (μg/g) as a function of temperature ( C) and modifier flow (ml/min), the other two factors (pressure and dynamic extraction time) being kept constant at their middle values. (200 bar), whereas for vindoline the pressure of 300 bar was more favourable [Fig. 5(A, B)]. Song et al. (1992) also obtained the highest recovery for vindoline at 300 bar. Since the same matrix contained both the alkaloids, therefore, the optimum pressure (250 bar) and temperature (80 C) were selected for carrying out replicate extractions (n = 6). Figure 6(A, B) show the interaction of temperature and pressure with the dynamic extraction time on the dry weight content of catharanthine. It can be seen that a maximum yield of catharanthine was obtained when the extractions were carried out for 40 min, both higher and lower extraction times showed a decrease in the dry weight content. For vindoline, a higher extraction time (60 min) was favourable when the interaction of pressure with dynamic extraction time was evaluated, whereas, a lower extraction time (20 min) gave higher yields when the interaction of temperature was evaluated. This could be due to possible degradation at higher temperatures with long extraction times. Therefore, the optimum time of 40 min was selected for the replicate extractions. Figure 7(A) shows the interaction of pressure and modifier flow on the yield of catharanthine. Higher dry weight contents were obtained with a modifier flow of 0.1 ml/min (6.6 vol%). Similar results were obtained for vindoline. The interaction of temperature and modifier flow gave a higher yield of vindoline at the lowest (0.04 ml/min) as well as at the highest (0.1 ml/min) modifier flow rates [Fig. 7(B)]. Since the flow rate of 0.1 ml/min was favourable for catharanthine as well, it was chosen as the optimum modifier flow. Comparison with traditional extraction methods The supercritical fluid extraction of Catharanthus alkaloids at optimised conditions was compared with traditional solid liquid extraction, Soxhlet extraction and hot water extraction at various temperatures. Six replicate experiments were carried out with SFE, whereas three replicates were performed with all other extraction methods. The relative recoveries of the three major alkaloids using different extraction methods are shown in Fig. 4. The best recoveries (100%) for catharanthine were obtained with SFE at optimised conditions, for vindoline with Soxhlet extraction using dichloromethane under reflux for 16 h, and that for 3,4 -anhydrovinblastine with traditional solid liquid extraction using a solution of 0.5 M sulphuric acid and methanol (3:1 v/v, ph 1.4) in an ultrasonic bath for 3 h. Dichloromethane has been used for the extraction of several types of alkaloids using different extraction methods. El Jaber-Vazdekis et al. (2006) claimed the higher extraction efficiency of dichloromethane and its replacement with chloroform. However, the use of chloroform in laboratories incurs major health, security and regulation problems. Although dichloromethane has also been suspected of being a toxicant to human organs, it is still widely used as a process solvent in the manufacturing of drugs and pharmaceuticals. It is a medium polar solvent generally suitable for the extraction of compounds with different polarities. Dichloromethane can provoke quaternisation of certain types of alkaloids (such as strychnine) producing crystalline precipitates at room temperature. The quaternisation

11 62 A. VERMA ET AL. of tropane alkaloids has been reported by Vincze and Gefen (1978) during MS analysis when high temperatures were used for sample volatilisation. We did not observe this phenomenon during the Soxhlet extraction of Catharanthus alkaloids using dichloromethane. Furthermore, if quaternisation had taken place, very polar compounds would have formed eluting much earlier than other major alkaloids, thus producing new peaks. No extra peaks were observed in the HPLC chromatogram of the dicholoromethane extract. The high recoveries of catharanthine (89.7%) and vindoline (100%) with Soxhlet extraction using dichloromethane as solvent could be due to the stability of these monomeric alkaloids at lower temperature (40 C, boiling point of dichloromethane). When methanol (boiling point 65 C) was used as solvent in the Soxhlet extraction catharanthine was completely decomposed after 16 h of extraction owing to its thermolabile nature. On the other hand, a ca. 48% recovery of catharanthine using hot water extraction for 3 h at 70 C was obtained, showing that this compound can be extracted at higher temperatures using shorter extraction times. Hot water extraction at 90 C resulted in the nearly complete decomposition of catharanthine and 3,4 -anhydrovinblastine. The recovery of vindoline ( %) remained unaffected by the increase in temperature during hot water extraction; vindoline seemed to be the most stable Catharanthus alkaloid. Both hydrochloric acid and sulphuric acid were compared for the extraction of major alkaloids. Extraction with sulphuric acid gave better results in comparison to hydrochloric acid. Hallard (2000) also showed that the relative extraction efficiency of vindolinine was better with sulphuric acid (50%) in comparison with hydrochloric acid (44%). The important result of this work was the extraction of 3,4 -anhydrovinblastine (38.4%) by SFE, although, its best recovery (100%) was obtained by the traditional solid liquid extraction using a solution of 0.5 M sulphuric acid and methanol (3:1 v/v, ph 1.4) in an ultrasonic bath for 3 h. This could be due to its improved solubility and stability at low ph, since the alkaloids are basic compounds and form salts easily in aqueous acidic medium, which enhances their solubility. In addition, the proton in aqueous acidic medium is probably more reactive and may break the sample matrix to release the analytes more easily in solid liquid extraction as compared with the inert supercritical carbon dioxide in SFE. The alkaloids in free base form were easily separated by liquid liquid extraction using organic solvents. We used methyl tert-butyl ether because it is a more environmentally friendly and non-toxic solvent in comparison with chloroform and dichloromethane and its extraction efficiency was similar to that of dichloromethane. High temperatures and longer extraction times also affected the recovery of 3,4 -anhydrovinblastine. Solid liquid extraction at room temperature for 3 h resulted in 100% relative recovery, whereas hot water extraction at C for 3 h gave only 9 2% recoveries. Soxhlet extraction with dichloromethane and with methanol for 16 h gave 18 and 0% recoveries, respectively. In SFE, the extractions were carried out at 80 C with a dynamic extraction time of 40 min, which resulted in 38% recovery. The above results clearly shows that 3,4 -anhydrovinblastine is a thermolabile compound and degrades easily if extracted at higher temperatures with longer extraction (or residence) times. The RSD for the major alkaloids varied from 4.1 to 17.5% with different extraction methods (Table 1). The RSD values for replicate extractions in SFE were from 6.5 to 7.2%, whereas in solid liquid extraction and hot water extraction the RSD values varied from 6.3 to 17.5%. The higher RSD percentages obtained with traditional extraction methods could be explained by the long and tedious sample preparation procedures employed, which included filtration, treatment with organic solvents, liquid liquid extraction, separation and evaporation steps, whereas in SFE the extraction took place in closed vessels and the collected extract was simply evaporated. In general, the extracts from SFE [Fig. 2(B)] were found to be much cleaner in comparison with those obtained by solid-liquid extractions (Fig. 3) or Soxhlet extractions. The results showed that supercritical fluid extraction is a valuable alternative technique to traditional extraction methods of Catharanthus alkaloids from dried leaves. To the best of our knowledge, the extraction of 3,4 -anhydrovinblastine and leurocine has not been reported earlier using SFE. The effect of four SFE factors on the dry weight content of catharanthine and vindoline was measured by applying a full factorial design and response surfaces were plotted to evaluate their interactions. Clearly the pressure and temperature were dominant factors in the extraction of Catharanthus alkaloids. Acknowledgements The authors wish to thank Matti Jussila for technical assistance in the use of ion-trap mass spectrometer (Bruker Daltonics). Financial support for this work was provided by the University of Helsinki (project ). REFERENCES Arens H, Stöckigt J, Weiler EW, Zenk MH Radioimmunoassays for the determination of the indole alkaloids ajmalicine and serpentine in plants. 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