61

Chapitre 4

Quantification of indole alkaloids and

iridoid precursors in Catharanthus roseus

hairy roots by high-performance liquid

chromatography

Cette partie présente le développement des méthodes HPLC utilisées pour déterminer les

concentrations en alcaloïdes de type indole et de leurs précurseurs dans les racines transfor-

mées deCatharanthus roseus.

Les 2 méthodes utilisent comme phase mobile de l’acétonitrile et un tampon de potassium

phosphate. Elles permettent la séparation et la quantification de la serpentine, vincristine, vin-

blastine, vindoline, catharanthine, tabersonine d’une part et du tryptophane, de la tryptamine,

secologanine et ajmalicine d’autre part.

Des racines transformées deC. roseusont été mises en culture en cuvée et élicitées par de

l’acide jasmonique (25 mg/l). La catharanthine, la serpentine, la tabersonine, la tryptamine, la

sécologanine et l’ajmalicine ont été quantifiées dans ces cultures. La vincristine, la vindoline,

la vinblastine et le tryptophane n’ont pas été détectés.

62

Des composées ayant un spectre UV proche de celui de la tabersonine ont été détectés. En

se basant sur les propriétés électroniques du recouvrement latéral des orbitales moléculaires

pz sur le squelette d’orbitales moléculaires hybrides sp2 de la tabersonine et de composés

proches de la tabersonine, l’hypothèse que les composés inconnus soient des dérivés de la

tabersonine est discutée.

Cette publication a été soumise à la revueJournal of Chromatography A.

63

MANUSCRIT # 1

Quantification of indole alkaloids and iridoid precursors in

Catharanthus roseushairy roots by high-performance liquid

chromatography

C. Tikhomiroff and M. Jolicoeur∗

Department of Chemical Engineering, Biopro Research Centre, École Polytechnique de

Montréal, P.O. Box 6079 Centre-ville Station, Montréal, Québec, Canada, H3C 3A7

∗ Corresponding author.

4.1 Abstract

Two direct HPLC analytical methods using photo diode array (PDA) and fluorescence

detection for the simultaneous quantification of the major indole alkaloids ofCatharan-

thus roseushairy roots and their iridoid precursors have been developed. The separation

of catharanthine, serpentine, tabersonine, vindoline, vinblastine, vincristine, ajmalicine, tryp-

tophane, tryptamine and secologanin was achieved on a reversed-phase C18 column. The

assays were successfully used to quantify the major indole alkaloids and their iridoid pre-

cursors in hairy root cultures ofC. roseus, thus providing a reliable tool for the study of the

secondary metabolism ofC. roseus. The use of PDA detection allowed for identification of

the compounds based on the retention time and the comparison of UV spectra with authentic

standards, as well as detection of five unknown compounds whose UV spectra are similar to

the tabersonine UV spectrum, suggesting that these compounds are tabersonine derivatives.

64

4.2 Key Words

Terpenoid Indole Alkaloids;Catharanthus roseushairy roots; HPLC

4.3 Introduction

Production of secondary metabolites by plant cells and tissues has become an active field

of study because of its potential as a source of new pharmaceutical compounds. In vitro

cultures of plant cells or tissues look promising for the large scale production of secondary

metabolites. Indeed, such cultures are not exposed to diseases and pests, and seem not to

be subject to seasonal and somatic variations.Catharanthus roseusproduces the anticancer

drugs vinblastine and vincristine, as well as the antihypertensive compounds ajmalicine and

serpentine (figure 4.1). Many studies have been conducted onC. roseuscell suspensions

but the absence of production of vindoline, a precursor of vinblastine and vincristine, has

always been reported [3,4]. The use ofAgrobacterium rhizogenestransformed hairy roots

cultures was similarly unsuccessful [3,5]. However, one study has reported the production of

vindoline in hairy roots ofC. roseus[6]. In contrast, ajmalicine and serpentine are always

detected [5,7,8]. Others indole alkaloids are found in these cultures, such as löchnericine and

hörhammericine. These compounds are believed to be tabersonine derivatives [7], but are un-

fortunately devoid of pharmaceutical value. One of our research interests focuses on the study

of C. roseushairy roots secondary metabolism under different environmental conditions, such

as elicitation, and nutritional states. A better understanding of the metabolic fluxes regulation

can be obtained with the metabolic flux analysis (MFA) method which requires measure-

ment of the biosynthesis reaction fluxes [9]. This implies the rapid quantification of indole

alkaloids for a large number of samples.

In order to measure the intracellular concentration of alkaloids, one has to extract, clean

and analyze the alkaloids from plant cells or hairy roots. The alkaline character of alkaloids is

often used for their isolation from plant material [10]. The extraction from lyophilized plant

is performed with methanol, ethyl acetate or chloroform. Soxhlet extraction can be used when

65

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66

a pre-separation is needed [11]. However, soxhlet extraction may alter the chemical nature

of the compounds under study and this procedure is somewhat time-consuming. Sonication

of the sample in methanol may be used instead of the soxhlet extraction [10]. The crude

plant extracts often need to be concentrated and fractioned in order to remove lipids, proteins,

pigments and other compounds. Once the samples have been purified, the separation and

quantification is performed. Thin layer chromatography (TLC) and colorimetry have been

used to isolate and quantify indole alkaloids ofC. roseus[10,12,13,14]. However, this method

is not suitable for routine analysis when high numbers of samples are involved. Moreover,

crude plant extract often contain many alkaloids and TLC is not always able to separate them

adequately.

Hight-Performance Liquid Chromatography (HPLC) systems equipped with an autosam-

pler provide a powerful tool to analyze large amounts of samples. The separation of indole

alkaloids is based on reversed phase chromatography using C18 as a stationary phase. Mobile

phases usually consist of a mixture of an organic phase (such as methanol or acetonitrile)

and a buffer solution such as n-heptane sulfonic acid [10,11], diammoniumphosphate [15],

or ammonium acetate supplemented with triethylamine [16]. Detection is performed using a

UV detector at fixed wavelength [11,16] or a fluorescence detector [15]. All these methods

allowed quantification of most of the indole alkaloids ofCatharanthus roseus. However, to

study the overall secondary metabolism ofCatharanthus roseus, the iridoid precursors such as

secologanin, loganine and the indole precursors such as trytophan and tryptamine also need to

be quantified. Recently, a novel HPLC method was proposed to quantify these compounds in

a crude extract ofC. roseus[17]. The indole alkaloid precursors were extracted in methanol.

This result suggested that one methanolic crude plant extract could be used to analyze both

indole alkaloids and their precursors. However, the overall procedure for quantification of in-

dole alkaloids remains quite complicated if all steps of extraction, concentration, purification

and analysis still have to be conducted.

This work presents the development of two HPLC methods that allow the quantification

of indole alkaloids and their precursors in two distinct runs of 30 minutes and 20 minutes.

These methods were optimized with a simplified sample preparation consisting of methanol

67

extraction from lyophilized biomass without purification and concentration in order to signif-

icantly shorten the sample preparation. The originality of the developed methods consists in

the simplification of the whole analysis procedure in order to rapidly analyze known terpenoid

indole alkaloids ofC. roseushairy roots in large numbers of samples.

4.4 Experimental

4.4.1 Chemicals

Small amounts of ajmalicine, serpentine, catharanthine, tabersonine and vindoline were

kindly provided by Dr. Archambault (Université de Trois-Rivières, Quebec, Canada). Sec-

ologanin (99% of purity) was purchased from Phytoconsult (Gorlaeus Laboratories, Leiden,

The Netherlands). Tryptamine, tryptophane, vincristine and vinblastine (98% of purity) were

purchased from Sigma-Aldrich (Oakville, Ontario, Canada).

4.4.2 Apparatus and chromatic conditions

The HPLC analysis was performed using a Beckman Coulter pump module 126, a Beck-

man Coulter auto-sampler model 508, a Beckman Coulter PDA detector 168 and a Jasco

model 821-FP fluorescence detector. A Zorbax Eclipse XDB-C18 4.6mm x 150 mm col-

umn (Hewlett Packard, Missisauga, Ontario, Canada), coupled with a Upchurch Scientific

4.3 mm x 1 cm ODS guard column (Upchurch Scientific, Concord, Ontario, Canada), was

used at a column temperature of 35◦C. The injection volume was 10µl. For the quantifi-

cation of catharanthine, serpentine, tabersonine, vindoline, vinblastine and vincristine, the

mobile phase consisted of a mixture of acetonitrile and 5 mM phosphate buffer (pH=6) with

a flow-rate of 2.0 ml/min. The eluent profile was: 0-20 minutes, linear gradient from 20:80

(v/v) to 80:20 (v/v); 20-25 minutes, isocratic elution with 80:20 (v/v) (column rinsing); 25-30

minutes isocratic elution with 20:80 (v/v) (column equilibration) (system I). For the quan-

tification of tryptophane, tryptamine, secologanin and ajmalicine, the method developed by

Dagnino et al. [17] was modified as follows: the mobile phase consisted of a 15:85 (v/v)

68

mixture of acetonitrile-100 mM phosphoric acid (pH=2) with a flow-rate of 1.8 ml/min. The

column was rinsed with a 85:15 (v/v) mobile phase for 5 minutes and equilibrated for 3 min-

utes (system II).

4.4.3 Standards solution

About 5 mg of ajmalicine, catharanthine, secologanin, tabersonine, tryptamine, vindoline,

vincristine and vinblastine were weighted on an analytical scale and individually dissolved

in methanol to a final volume of 1 ml. Same quantities of tryptophan and serpentine were

individually dissolved in a solution of MeOH:H2O (50:50, v/v) to a final volume of 1 ml.

These stock solutions were stored at -20◦C. The quantification was performed using 6 levels

of external standards. The more concentrated standard used for quantification consisted of a

dilution of 30 µl of each stock solution into 1.40 ml of methanol. The five other standards

were obtained by dilution by 2,22, 23, 24, and25 of the latter standard.

4.4.4 Sample preparation

Approximately 200 mg of fresh hairy roots were lyophilized overnight. The dry roots

were weighted, crushed in a tissue grinder (VWR Canlab, Ville Mont-royal, Québec,

CANADA) and extracted in 1 ml of MeOH for 60 minutes in a sonicating bath. The ex-

tract was centrifuged and the supernatant was filtered through a PTFE 0.45µm filter prior to

injection in the HPLC system.

4.4.5 Extraction recovery, peak identification, peak purity and limits of

detection

Alkaloids and their precursors were extracted from lyophilized hairy roots ofC. roseus

with or without known amounts of standards (approximately 4 mg per g dry weight of hairy

root) and quantified to evaluate the extraction recovery of each compound. For all alkaloids,

both UV spectra and retention time of spiked samples were identical to those of the standards,

69

suggesting that no matrix effect was present. Identification of alkaloids from the crude plant

extract was established by comparison of the UV spectra and retention time with authentic

standards. The purity of peaks was based on the calculation of the ratio of absorbances at 220

and 280 nm compared to that of standards [18,19]. Limit of detection were determined when

the ratio of the standard’s peak area to noise was greater than five.

4.4.6 Hairy root cultures of Catharanthus roseus

C. roseusL.G. Don hairy roots were established as already described [11]. Root cultures

were grown in Petri dishes on minimum medium [20] supplemented with 3% (w/v) sucrose,

as well as a tenfold KH2PO4 and a threefold Ca(NO3)2 concentration. They were elicited

using 25 mg/l jasmonic acid [5] during the exponential growth phase and harvested three

days later for quantification of indole alkaloids and precursors.

4.5 Results and Discussion

4.5.1 Separation of indole alkaloids and precursors

For the separation of alkaloids (system I), the studies were conducted with a mixture of aj-

malicine, serpentine, catharanthine, tabersonine, vindoline, vinblastine and vincristine whose

individual UV spectra were first characterized (figure 4.2). Optimal chromatographic condi-

tions were obtained after testing different mobile phases with a reversed-phase C18 column.

All mobile phases consisted of an organic solvent (methanol, acetonitrile and acetonitrile +

0.05-0.1% (v/v) methanol) and potassium phosphate buffer (5 mM) with pH of 2, 4, 6 and

8. Isocratic elutions were performed but unsatisfactory alkaloid separations were obtained or

run time was long (more than 1 hour). Linear gradient elutions were investigated and separa-

tion of most of the alkaloids was possible during a 60 minutes run at a flow-rate of 1 mL/min.

Catharanthine, vindoline, vincristine and vinblastine poorly separated. This behavior did not

depend upon the pH of the phosphate buffer. In contrast, ajmalicine coeluted with catharan-

thine at pH=8 and serpentine coeluted with catharanthine at any pH below 6. Tabersonine was

70

Figure 4.2: UV spectrum of studied alkaloids and precursors. A, catharanthine; B, serpen-tine; C, tabersonine; D, vinblastine; E, vincristine; F, vindoline; G, ajmalicine; H tryptophane;I, tryptamine; J, secologanin.

71

always the last eluted compound and was easy to separate from the other alkaloids. The best

results were obtained with a mobile phase of acetonitrile:potassium phosphate buffer (pH=6)

with the following elution profile: linear gradient from 20:80 (v/v) to 80:20 in 40 minutes,

isocratic elution at 80:20 for 10 minutes. The total run time including 10 minutes of column

equilibration was 60 minutes. The flow was raised to 2 ml/min and the gradient elution was

conducted in 20 minutes. The separation was found to be the same than at a flow-rate of

1 ml/min, but twice as fast. The results demonstrated an excellent separation of the seven

external standards (figure 4.3). The detection of alkaloids was done with a PDA using the 3D

mode, allowing to collect the UV spectra of the eluent in a wavelength range from 210 to 360

nm by steps of 2 nm in real time. As shown on figure 4.2 catharanthine, vindoline, vinblas-

tine and vincristine have a high absorbance at 220m, tabersonine at 330 nm, ajmalicine at 210

nm and serpentine at 250 nm. These UV spectra were used to select the best UV detection

conditions.

For the separation of alkaloids precursors (system II), the method developed by Dagnino,

et al. [17] was modified to allow separation of tryptophane, tryptamine, secologanin and

ajmalicine. Ajmalicine eluted after secologanin, as shown in figure 4.4. The four com-

pounds could be separated in less than 13 minutes. UV spectrum of each compound was

recorded (figure 4.2). Secologanin was detected at 238 nm and ajmalicine at 210 nm. Al-

though tryptamine and tryptophane could be detected at 218 nm, their detection by fluores-

cence at an excitation wavelength of 270 nm and an emission wavelength of 370 nm provided

a higher sensibility of detection.

4.5.2 Analysis of a crude plant extract

A methanolic extract ofC. roseushairy roots was injected to evaluate the separation effi-

ciency of the alkaloids. With the system I, ajmalicine and serpentine coeluted with unknown

compounds that have a high absorbance between 210 and 250 nm. As the maximum ab-

sorbance wavelength of ajmalicine was 210 nm, its quantification was not possible. Serpen-

tine has at least two maximum absorbance wavelengths (250 and 306) nm. The unknown

72

Figure 4.3: HPLC chromatogram of pure indole alkaloids at 210 nm. 1, ajmalicine; 2,serpentine; 3, vincristine; 4, vindoline; 5, catharanthine; 6, vinblastine; 7, tabersonine.

Figure 4.4: HPLC chromatogram at 238 nm of pure indole alkaloids precursors. 1, trypto-phane; 2, tryptamine; 3, secologanin; 4, ajmalicine.

73

compound did not interfere with serpentine at 306 nm, so it was possible to quantify serpen-

tine at this wavelength. Catharanthine and tabersonine were detected as pure compounds. The

figure 4.5 presents the chromatograms of the crude extract. However, as reported previously

[4], vincristine, vindoline and vinblastine were not detected in hairy root cultures ofC. roseus

of the present study. These results gave us the final detection parameters for the quantification

of serpentine, catharanthine, vindoline, vincristine and vinblastine as shown in table 4.1. The

system II was able to separate tryptamine, secologanin and ajmalicine from the same crude

extract as pure compounds. However, only traces of tryptophane were detected in all assays

(figure 4.6) and no other compound was detected at the tryptophan retention time. Thus, the

final parameters for the detection of ajmalicine, tryptophan, tryptamine and secologanin were

those determined above. The calibration curves exhibited linear regression (r > 0.997) for

both HPLC systems.

The extraction recovery is not the same for all alkaloids. Ajmalicine and secologanin

are completely extracted from the hairy roots whereas catharanthine, serpentine, tabersonine,

vindoline and vincristine are partially extracted (table 4.1). Although the proposed extraction

method does not have a 100% extraction recovery yield for all studied compounds, it is ap-

propriate to quantify indole alkaloids and its precursors in hairy roots ofC. roseus. Internal

standards should be used when high accuracy is required.

4.5.3 Unknown compounds of interest

The chromatograms of crude extract ofC. roseusexhibited 5 unknown compounds (T1-

T5) that have a UV spectrum quite similar to that of tabersonine (figure 4.7) but have different

retention times: 11.2 min (3), 16.5 min (4), 19.2 min (5), 22.3 min (7) (figure 4.5), and 4.2 min

(3) (figure 4.6). Tabersonine is known to yield to vindoline through five intermediary com-

pounds: 16-hydroxytabersonine, 16-methoxytabersonine, 16-methoxy-2,3-dihydro-3-hydro-

xytabersonine, deacetoxyvindoline, and deacetylvindoine [2,and references therein]. Alter-

native pathways for the degradation of tabersonine have been established [7]. Löchnericine

and hörhammericine are derived from tabersonine by epoxydation of the 6-7 bond. Moreover,

74

Figure 4.5: HPLC chromatograms ofC. roseushairy root extract at 220 nm (A), 306 nm (B)and 330 nm (C). 1, catharanthine; 2, serpentine; 3, unknown T1; 4, unknown T2; 5, unknownT3; 6, tabersonine; 7, unknown T4.

75

Figure 4.6: HPLC chromatograms of aC. roseushairy root extract: fluorescence response at270 nm excitation and 370 nm emission (A). UV absorbance at 210 nm (B) and 238 nm (C).1, tryptophane; 2, tryptamine; 3, unknown T5; 4, ajmalicine; 5, secologanin.

76

Table 4.1: Detection parameters of indole alkaloids and precursors (HPLC systems I and II)

Metabolite Retentiontime (min)

Wavelength(nm)

Test range(µg/ml)

Limitof de-tection(µg/ml)

Extractionyield (%)a

Serpentine 7.8 306 3.0-96.1 0.884 32.4±4

Vincristine 13.1 220 2.68-85.8 0.790 43.2±10

Vindoline 13.5 220 3.05-97.6 0.898 39.2±3

Catharanthine 14.5 220 2.84-90.8 0.835 24.6±2

Vinblastine 15.2 220 3.7-118 1.09 88±16

Tabersonine 20.8 330 2.72-87.1 0.201 34.5±1

Tryptophane 3.2 270/370b 1.77-56.6 0.110 54.8±8

Tryptamine 3.5 270/370b 1.52-48.5 0.095 85±23

Secologanin 7.2 238 2.73-87.4 1.36 110±5

Ajmalicine 12.2 210 3.63-116 1.81 126±6

a Intervals are standard deviation (n = 3)b Fluorescence detection at excitation/emission wavelength

hörhammericine is thought to be synthesized from löchnericine or from tabersonine with the

formation of 19-hydroxytabersonine as an intermediary compound [1].

All these tabersonine derivatives except for the three preceding vindoline biosynthesis

share the same delocalizedπ-system on carbons 2, 3, 13, 14, 15, 16, 17, 18, 21 and on nitrogen

1 through a network ofpz molecular orbitals. The commonπ-system is presented with bold

bonds in the figure 4.8. Such an expanded delocalizedπ-system is usually responsible for

absorption in the low UV and visible range. The particular UV spectrum of tabersonine could

be due to this topology. With this hypothesis, the five unknown compounds T1 to T5 detected

in our hairy root cultures could be some of the five tabersonine derivatives having the outlined

π-bond (löchnericine, hörhammericine, 16-hydroxytabersonine, 16-methoxytabersonine and

19-hydroxytabersonine). These unknown compounds will be identified by MS and NMR to

confirm this hypothesis.

77

Figure 4.7: UV spectra of tabersonine compared to unknown compounds T1 to T5.

78

Figure 4.8: Chemical structure of tabersonine derivatives [1,2]. The bold lines stand forbonds participating in the sameπ-system. 16-methoxy-2,3-dihydro3-hydroxytabersonine andall following precursors of vindoline have a differentπ-system.

79

4.6 Conclusion

Two effective HPLC reversed-phase HPLC methods were developed that allowed separa-

tion of manyC. roseusindole alkaloids, as well as some of the iridoids and indole precursors

in C. roseuscrude extracts. Catharanthine, serpentine, tabersonine, ajmalicine, secologanin

and tryptamine were successfully quantified in elicited hairy root cultures, whereas vindo-

line, vincristine, vinblastine and tryptophane were not detected. Excellent separation of the

standards persisted in the crude plant extracts despite the simplification of the extraction pro-

cedure. Theses methods allowed the separation of unknown compounds among which five

are thought to be tabersonine derivatives. The rapid quantification of many alkaloids and pre-

cursors will enable future studies on the calculation of metabolic flux of the terpenoid indole

alkaloids ofC. roseushairy roots.

4.7 Acknowledgements

C. Tikhomiroff has a fellowship from the Fondation de l’École Polytechnique (France).

The HPLC system was purchased from a grant from the NSERC. The FCAR funded this

research project. The authors wish to thanks M. Klvana and N. Chauret for reviewing this

document.

4.8 References

[1] Morgan, J. A., Shanks, J.V., Phytochem., 51 (1999) 61-68

[2] De Luca, V., Laflamme, P., Cur. Opinion Plant Biol., 4 (2001) 225

[3] van der Heijden, R., Verpoorte, R., ten Hoopen, H. J. G., Plant Cell Tiss. Organ Cult.,

18 (1989) 231

[4] Moreno, P. R. H., van der Haiden, R., Verpoorte, R., Organ Cult., 42 (1995) 1

[5] Rijhwani, S. K., Shanks, J.V., Biotechnol. Prog., 14 (1998) 442

80

[6] Parr, A.J., Peerless, A.C.J., Hamill, J.D., Walton, N.J., Robins, R.J., Rhodes, M.J.C.,

Plant Cell Rep., 7 (1988) 309

[7] Shanks, J. V., Rajiv, R., Morgan, J., Rijhawani, S., Vani, S., Biotechnol. Bioeng.,

58(2&3) (1997) 333

[8] Bhadra, R., Shanks, J.V., Biotechnol. Bioeng., 55(3) (1997) 527

[9] Stephanopoulos, G.N., Aristidou, A.A., Nielsen, J., Metabolic Engineering,

Academic Press, San Diego, 1998

[10] Morris, P., Scragg, A.H., Smart, N.J., Stafford, A., in Plant Cell Culture, a practical

approach, RL Press, Oxford, 1985, Ch. 7 p. 127

[11] Bhadra, R., Vani, S. and Shanks, J. V., Biotechnol. Bioeng., 41(5) (1993) 581

[12] Vazquez-Flota, F., Moreno-Valenzuela, O., Miranda-Ham, Coello-Coello-J.,

Loyola-Vargas, V. M., Plant Cell. Tissue Organ Cult., 38 (1994) 273

[13] Montforte-Gonzalez, M., Ayora-Talavera, T., Maldonado-Mendoza, I.E.,

Loyola-Vargas V.M., Phtytochem. Ana., 3 (1992) 117

[14] Farnsworthm N.R., B., R.N, Damratoski, D., Meer, W.A., Camparato, L.V., Lloydia,

27 (1964) 302

[15] Renaudin, J.-P., Physiol. Vég., 23(4) (1985) 381

[16] Toivonen, L., Ojala. M., Kauppinen, V., Biotechnol. Bioeng., 37 (1991) 673

[17] Dagnino, D., Schripsema, J., Verpoorte, R., Planta Med., 62 (1995) 2789

[18] Cheng, H., Gadde, R.R., J Chromatogr. Sci, 23 (1985) 227

[19] Fell, A.F., Scott, H.P., J Chromatogr. A, 273 (1983) 3

[20] Bécard, G. and Fortin, J. A., New Phytol., 108 (1988) 211