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Extraction of Indole Alkaloids from Catharanthus roseus Using

Supercritical Carbon Dioxide

Article  in  Biotechnology Techniques · March 1992

DOI: 10.1007/BF02438817

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 BIOTECHNOLOGY TECHNIQUES
Vohtme 6 No.2 marctdapri11992 pp.127-130
Received 4th December

EXTRACTION OF INDOLE ALKALOIDS FROM CATHARANTHUS

ROSEUS BY USING SUPERCRITICAL CARBON DIOXIDE

Huen Lee1., Won-Hi Hong1, Ji-Ho Yoon1, Kyu-Min Song1,

Sang-Soo Kwak2, and Jang-Ryol Liu2
1) BioProcess Engineering Center, KAIST, 373-1,
Kusung-dong, Yusung-gu, Taejon, 305-701, Korea
2) Plant Cell Biology Lab., Genetic Engineering Research Institute, KIST,
P.O. Box 17, Taedok Science Town, Taejon, Korea

Summary

Indole alkaloids, particularly vindoline and catharanthine, were extracted from the leaves of
Catharanthus roseus by supercritical extraction with CO2. The contents of vindoline and
catharanthine in the extracts were determined by HPLC and identified by LC/MS. About 52
%(w/w) of the initial vindoline content, 1.5 mg vindoline/g dry wt leaves, was recovered after
extracting this material for 10 h with the C O 2 flOW rate of 400 ml/min at 40~ and 150 bar.
Vindoline concentration in the extract was 67 %(w/w).

Introduction

The medicinal plant Catharanthus roseus, Madagascan periwincle, contains various indole
alkaloids of which vindoline and catharanthine are the most important. Vindoline and
catharanthine are well known as the precursors in the biosynthetic pathways of valuable dimerie
indole alkaloids such as vinblastine and vincristine. These two dimeric alkaloids have been used
for many years as chemotherapeutic agents in the treatment of acute leukemia and Hodgkin's
disease (Svoboda et al., 1959; Boder et al., 1964; Miura et al., 1988). However, bulk production
of vindoline has not been successful by plant cell tissue cultures although catharanthine has
been so produced (Drapeau et al., 1987; Smith et al., 1987; Tallevi and DiCosmo, 1988). In
addition, as vindoline content in the intact plant is quite small, its isolation by column
chromatography on a large scale is tedious and time-consuming. In this connection the objective
of this study was to test the applicability of supercritical fluid extraction (SFE) process for
selectively extracting vindoline from the leaves of Catharanthus roseus by using carbon dioxide
as a supercritical solvent.

127
Materials and Methods

Sample Preparation

The leaves of C a t h a r a n t h u s roseus were dried for 2413 in the oven maintained at 60~ and
then powdered to 200-300 mesh size in order to increase the mass transfer efficiency between
supercritical carbon dioxide and the sample particles.

SFE Procedure

A continuous flow-through SFE system was used for the tests. Carbon dioxide was supplied
from a gas cylinder and was directed to an electrically driven diaphragm-type compressor. It
was filtered through 10 a m filter-discs. After compression, the carbon dioxide was introduced
into a surge vessel to dampen the fluctuations generated by the compressor. To maintain a
constant pressure within the system, a back pressure regulator with a accuracy of + 1 % of the
relief pressure range was employed. The extraction column, 100em3, was packed with 3-ram
glass beads and about 7g powdered Catharanthus roseus in order to disperse the solvent flow
and to minimize the pressure drop through the column less than 1 bar. A metal filter was
inserted at the top of the column to prevent the entrainment of fine sample particles during
the experiment. The temperature inside the air bath was controlled to within _+0.1*e by using
a proportional temperature controller. The carbon dioxide-solute mixture leaving the top of
the extractor was expanded to atmospheric pressure through a micrometering valve into cold
traps where the solute has condensed. The flow rate and volume of carbon dioxide were measured
by a flow meter and a dry test meter. The dry test meter was equipped with gauges to measure
flow temperature and pressure. The use of low solvent flow rates guarantees the attainment
of equilibrium conditions at the extractor's outlet.

Quantitative Analysis by HPLC

The crude alkaloid extract obtained from the SFE experiment was analysed by a Hitachi
655A-12 HPLC. The extract was dissolved in a small volume of methanol and filtered through
a 0.5/.tm FH-type Millipore filter. This sample was then loaded onto a reversed-phase /~-
Bondapack C_as column(30em x 3.9m). The run was performed using 5raM (NH4)2PO 4
(pH 7.3) / methanol / acetonitrile (3:4:3) with a flow rate of 1 me/rain. The compounds were
detected by absorbance a t 2 9 8 nm. The quantitative analysis was determined by comparing
the peak areas of the sample with those of authentic alkaloids.

Identification by LC/MS

The contents of vindoline and catharanthine in the extracts were also checked by a Hewlett-
Packard 5988A mass spectrometer equipped with a particle-beam LC/MS interface. The solvent
for LC/MS was eluted with methanol / acetonitrile / 5 mM ammonium acetate(3:4:3) at a flow
rate of 0.5 me/min. The,other conditions for LC were the same as described in the quantitative
analysis. Electron impact ionization at 300~ temperature and with 278 /.tA current and 70
eV voltage was used for the MS.

Results and Discussion

The extraction of vindoline and catharanthine from C a t h a r a n t h u s r o s e u s was carried out

at several temperature and pressure conditions by using carbon dioxide as a supercritical solvent
and the contents of the extracts analysed by HPLC are summarized in Table 1.

128
 III'5988A MS
with Particle-Beam I.CIMS Interface
2.5E41 vi ride I i no"-+ cal haranl hint Electron voltage : 70 eV
Electron current : Z78 ~ A
2.8E41 A i o n i z a t i o n mode : El ( E l e c t r o n Impact)
Mass s c a n r a n g e : 70 - 500 amu

#: 58881
10 28 30 40 50
Time (mln,)

2.5E4
B
t35 /
e2.8E4 / 188 c.# ~"v'-? .TA.~,,
oOCOC"3
o CI!3 (5OOCH3
=1.5E4 187
3139 vindoline
"a10088 398

J ~,I,bl,Ill \
e--

222 X
8 ! i , ,),, , J I, | ! J
Ii
188 288 388
Ha.ss/Ch a.rge

2588" tat /
q.)
288(3- 135 COOCH3
t,,J
t"-
1588- c I i68 catharat~thine

¢,.,
1888- \
500i I 229
/
108 15G 208 258 388
H&ss/Ch &rge

F i g u r e 1. LC/MS analysis of vindoline and c a t h a r a n t h i n e extracted f r o m leaves of

Catharanthus roseus by using supercritical carbon dioxide.
A. Total ion c h r o m a t o g r a m of c r u d e alkaloids.
B. Mass s p e c t r u m of vindoline identified.
C. Mass s p e c t r u m of c a t h a r a n t h i n e identified.

129
 Table 1, Concentrations of vindoline and catharanthine in the extracts at five different
temperature and pressure conditions.

Vindoline % Catharanthine %
T('C) P(bar)
in extract in extract

40 150 67.2 trace

350 61.3 5.8
400 46.8 10.6
70 150 64.1 5.5
400 47.1 8.2

The qualitative identification of vindoline and eatharanthine was confirmed by LC/MS

(Figure 1). About 52 %(w/w) of the initial vindoline content, 1.5 mg vindoline/g dry wt leaves,
was recovered after extracting this material for 10 h with the CO2 flow rate of 400 ml/min
at 40~ and 150 bar. Vindoline concentration in the extract was 67 %(w/w). The selectivity
of SC-CO2 for vindoline was decreased with pressure at constant temperature, whereas for
catharanthine increased with pressure (Table 1). However, the effect of temperature on the
selectivity was quite small for both indole alkaloids. The high selectivity of SC-CO2 for vindoline,
i.e., 67 %(w/w) at .40~ and 150 bar, shows that the SFE process would provide a potential
route for bulk production of vindoline from Catharanthus roseus. However, in our opinion these
overall results stated above are preliminary and further investigations are needed to resolve
some essential problems such as the interaction between supercritical solvent and various
alkaloids, the enhancement of both solvent power and selectivity by a small addition of
cosolvents, the determination of optimal SFE conditions and finally many other factors related
to commercial scale-up.

Acknowledgment

This work was supported by the Ministry of Science and Technology and the Korea Science
and Engineering Foundation.

REFERENCES

Boder, G.B., Gorman, M., Johnson, I.S., and Simpson, P.J. (1964). Lloydia 27, 328-333.
Drapeau, D., Blanch, H.W., and Wilke, C.R. (1987). Planta Med., 53, 373-376.
Miura, Y., Hirata, K., Kurando, N., Miyamoto, K., and Uchida, K.(1988). Planta Med. 54,
18-20.
Smith, J.I., Smart, N.J., Kurz, W.G.W., and Misawa, M.(1987). Planta Med. 53, 470-474.
Svoboda, G.H., Neuss, N., and Gorman, M.(1959). J. Am. Pharm. Assoc. 48, 659-666.
Tallevi, S.G. and DiCosmo, F.(1988). Planta Med. 54, 149-152.

130

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